Method for the production of p-hydroxybenzoate in species of Pseudomonas and Agrobacterium

ABSTRACT

Bacterial strains transformed with the pcu genes are useful for the production of para-hydroxybenzoate (PHBA). Applicant has provided the p-cresol utilizing (pcu) and tmoX gene sequences from  Pseudomonas mendocina  KR-1, the proteins encoded by these sequences, recombinant plasmids containing such sequences, and bacterial host cells containing such plasmids or integrated sequences. Method for the use of these materials to produce PHBA are also disclosed.

FIELD OF INVENTION

[0001] The present invention relates to the fields of molecular biologyand microbiology, and to the use of genetic techniques to introduce amodified pathway for the production of desired compounds. Morespecifically, this invention describes genetically engineeredbiocatalysts possessing an enhanced, or new, ability to transformp-cresol or toluene to p-hydroxybenzoate.

BACKGROUND OF THE INVENTION

[0002] p-Hydroxybenzoate (PHBA) is used as a monomer for synthesizingLiquid Crystal Polymers (LCP). LCP's are used in electronic connectorsand in telecommunication and aerospace applications. LCP resistance tosterilizing radiation suits these materials for use in medical devicesas well as in chemical, and food packaging applications. Esters of PHBAalso are used as backbone modifiers in other condensation polymers(i.e., polyesters), and are also used to make parabens preservatives.

[0003] Chemical synthesis of PHBA is known. For example, JP 05009154teaches a chemical route using the Kolbe-Schmidt process from tar acidand CO₂ involving 1) the extraction of tar acid from a tar naphthaleneoil by an aqueous potassium hydroxide, 2) adding phenol to the extractedtar acid potassium salt, 3) removing H₂O, and 4) reacting the resultantslurry with CO₂. Alternative methods of chemical synthesis are known(see, for example, U.S. Pat. Nos. 5,399,178; 4,740,614; and 3,985,797).

[0004] However, chemical synthesis of PHBA is problematic and costly dueto the high energy needed for synthesis and the extensive purificationof product required. An alternate low cost method with simplifiedpurification would represent an advance in the art. Biologicalproduction offers one such low cost, simplified solution to thisproblem.

[0005] Microbiological methods of PHBA synthesis are known. For example,JP 06078780 teaches PHBA preparation by culturing benzoic acid in thepresence of microorganisms (preferably Aspergillus) that oxidize benzoicacid to PHBA.

[0006] An alternate method of biological production is suggested bybacteria that have an enzymatic pathway for the degradation of tolueneand other organics where PHBA is produced as an intermediate. The firstenzyme in the toluene degradation pathway is toluene monooxygenase (TMO)and the pathway is referred to as the TMO pathway. The steps of the TMOpathway have been described (Whited and Gibson, J. Bacteriol.173:3010-3020 (1991)) and are illustrated in FIG. 1. Bacteria thatpossess the toluene degradation pathway are found in the genusPseudomonas where Pseudomonas putida, Pseudomonas fluorescens,Pseudomonas aeruginosa and Pseudomonas mendocina are the most commonlyutilized species. Other examples of aerobic bacteria that are known todegrade toluene are Burkholderia (Johnson et al., Appl. Environ.Microbiol. 63:4047-4052 (1997)), Mycobacterium (Stephen et al., Appl.Environ. Microbiol. 64:1715-1720 (1998)), Sphingomonas (Zylstra et al.,J. Ind. Microbiol Biotechnol. 19:408-414 (1997)) and Rhodococcus (Kosonoet al., Appl. Environ. Microbiol. 63:3282-3285 (1997)). In addition,several different species of anaerobic bacteria are known to utilizetoluene (Heider et al., Anarobe 3:1-22 (1997)). Toluene degradationpathways have been highly characterized (Romine et al., InBioremediation of Chlorinated Polycyclic Aromatic Hydrocarbon Compounds;Hinchee, R. E., Ed.; Lewis: Boca Raton, Fla., 1994; pp 271-276) and anumber of the genes encoding key enzymes have been cloned and sequenced,including the protocatechuate 3,4-dioxygenase genes (Frazee, J.Bacteriol. 175(19):6194-6202 (1993)), the pcaR regulatory gene fromPseudomonas putida, which is required for the complete degradation ofp-hydroxybenzoate (Romero-Steiner et al., J. Bacteriol.176(18):5771-5779 (1994); Dimarco et al., J. Bacteriol.176(14):4277-4284 (1994)) and the pobA gene encoding the expression ofp-hydroxybenzoate hydroxylase (PHBH), the principal enzyme for theconversion of PHBA to protocatechuate (Wong et al., Microbiology(Reading U.K.) 140(10):2775-2786 (1994); Entsch et al., Gene71(2):279-291 (1988)).

[0007] Bacteria that possess the TMO pathway are useful for degradingtoluene and trichloroethylene. They are able to use these and otherorganics as sole carbon sources where they are transformed through PHBAto ring-opening degradation products (U.S. Pat. Nos. 5,017,495;5,079,166; 4,910,143). By using the chromosomal TMO pathway, incombination with mutations that prevent PHBA degradation in Pseudomonasmendocina KR1, it has been shown that PHBA can be accumulated byoxidation of toluene (PCT/US98/12072).

[0008] Recently, various strains of Pseudomonas possessing the toluenedegradation pathways have been used to produce muconic acid viamanipulation of growth conditions (U.S. Pat. Nos. 4,657,863; 4,968,612).Additionally, strains of Enterobacter with the ability to convertp-cresol to PHBA have been isolated from soil (JP 05328981). Further, JP05336980 and JP 05336979 disclose isolated strains of Pseudomonas putidawith the ability to produce PHBA from p-cresol. Additionly, Miller andcoworkers (Green Chem. 1(3):143-152 (1999)) have shown the bioconversionof toluene to PHBA via the construction of a recombinant Pseudomonasputida. Their initial catalyst development focused on Pseudomonasmendocina KR1 for production of PHBA from toluene. However, they wereunable to obtain significant accumulation of PHBA from toluene usingthis strain. This result was due to their inability to obtain asufficient disruption of PobA activity (the enzyme catalyzingm-hydroxylation of PHBA to protocatechuate in the protocatechuate branchof the β-ketoadipate pathway; see FIG. 1).

[0009] Although the presence of the TMO pathway in Pseudomonas mendocinaKR1 has been documented (Wright and Olsen, Applied Environ. Microbiol.60(1):235-242 (1994)), the art has not provided a molecularcharacterization and sequence of the pcu genes encoding the enzymes thattransform p-cresol to PHBA in this organism. The art has also notprovided bacterial host cells harboring novel recombinant plasmidsencoding the enzymes of p-cresol to PHBA oxidation, together withoperably-linked native promoter and regulatory sequences and proteins.Such bacterial host strains, if they lack the enzymes to degrade PHBAfurther, can accumulate PHBA when cultured in the presence of p-cresol.

[0010] As an alternative to culturing cells in the presence of p-cresol,the latter compound can be formed from toluene in cells thatadditionally harbor plasmid-encoded toluene monooxygenase. A bacterialstrain harboring plasmid-encoded tmo and pcu operons has not been fullydescribed in the art, particularly a strain that exceeds the productionlevel of PHBA when compared to plasmid-free Pseudomonas mendocina KR1.In addition, expression of the tmo operon using its nativetoluene-induced promoter localized upstream of a tmoX gene previouslyhas not been known. Therefore, the problem to be solved is the lack of afully characterized pcu operon and the availability of a bacterialstrain harboring plasmid-encoded tmo and pcu operons to use for thebioproduction of PHBA.

SUMMARY OF THE INVENTION

[0011] The present invention solves the problem of extensivelycharacterizing the pcu operon by providing cloned, sequenced, andexpressed genes of the pcu operon from Pseudomonas mendocina KR-1 thatcan be transformed into and used to produce PHBA from p-cresol inPseudomonas putida and Agrobacterium rhizogenes strains that do notnormally possess this capability. In addition, transformation of the pcuoperon into Pseudomonas mendocina KRC16KDpobA51 supplements theendogenous pcu operon leading to an increase in PHBA production. Thisincrease in PHBA production in Pseudomonas mendocina KRC16KDpobA51transformed with plasmid-encoded pcu is an improvement overPCT/US98/12072.

[0012] The present invention provides a method for the production ofPHBA comprising: (i) culturing a Pseudomonas, Agrobacterium or relatedstrain transformed with a pcu operon in a medium containing an aromaticorganic substrate, at least one suitable-fermentable carbon source, anda nitrogen source, wherein the supplied pcu operon comprises genesencoding the TMO toluene degradation pathway enzymes p-cresolmethylhydroxylase and p-hydroxy-benzaldehyde dehydrogenase, thetranscriptional activator PcuR, wherein the transformed Pseudomonas orAgrobacterium strain does not produce any detectable p-hydroxybenzoatehydroxylase, whereby PHBA accumulates; and (ii) recovering the PHBA.

[0013] The present invention also encompasses the combination of the pcuand tmo operons on a single replicon such that expression of tmo isobtained by transcription from a previously undisclosed toluene orp-cresol induced tmoX promoter, and expression of pcu is obtained bytranscription using a previously undisclosed sequence encoding atranscriptional activator.

[0014] Another preferred embodiment of the present invention includesthe recombinant plasmid pMC4 in Pseudomonas putida DOT-T1. This strainsynthesized the highest levels of tmo and pcu-encoded enzymes observedand is described herein.

[0015] It has also been found that the heterologous todST proteins thatcontrol the induction of toluene dioxygenase pathway induce high levelsof expression from the tmo pathway genes, and are useful tools tomediate expression of the catabolic tmo genes and PHBA production in anyorganism that does not possess these genes.

BRIEF DESCRIPTION OF THE DRAWINGS SE7QUENCE DESCRIPTIONS, AND BIOLOGICALDEPOSITS

[0016] The invention can be more fully understood with reference to thedrawings, from the detailed description, and the sequence descriptionswhich form part of this application.

[0017]FIG. 1 illustrates the pathway of the toluene degradation inPseudomonas mendocina KR-1.

[0018]FIG. 2 illustrates Pseudomonas mendocina KR-1 pcu operon.

[0019]FIG. 3 illustrates Pseudomonas mendocina KR-1 tmo operon.

[0020]FIG. 4 illustrates the pcu and tmo expression plasmid pMC4.

[0021] The following 112 sequence descriptions contained in thesequences listing attached hereto comply with the rules governingnucleotide and/or amino acid sequence disclosures in patent applicationsas set forth in 37 C.F.R. §1.821-1.825 (“Requirements for PatentApplications Containing Nucleotide Sequences and/or Amino Acid SequenceDisclosures—the Sequence Rules”) and are consistent with WorldIntellectual Property Organization (WIPO) Standard ST2.5 (1998) and thesequence listing requirements of the EPO and PCT (Rules 5.2 and49.5(a-bis), and-Section-208 and Annex C of the AdministrationInstructions). The Sequence Descriptions contain the one letter code fornucleotide sequence characters and the three letter codes for aminoacids as defined in conformity with the IUPAC-IYUB standards describedin Nucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical Journal219:345-373 (1984) which are herein incorporated by reference.

[0022] SEQ ID NO:1 is the nucleotide sequence of the pcu operon isolatedfrom Pseudomonas mendocina KR-1 (6491 bp).

[0023] SEQ ID NO:2 is the deduced amino acid sequence of thetranscriptional activator PcuR encoded by ORF1.1 (SEQ ID NO:98).

[0024] SEQ ID NO:3 is the deduced amino acid sequence of PcuC encoded byORF1.2 (SEQ ID NO:99) which has the enzyme activity of PHBAD.

[0025] SEQ ID NO:4 is the deduced amino acid sequence of PcuA encoded byORF1.3 (SEQ ID NO:100) which has the enzyme activity of PCMH.

[0026] SEQ ID NO:5 is the deduced amino acid sequence of PcuX encoded byORF1.4 (SEQ ID NO:101) which is an unidentified open reading frame andwhich may be an inner membrane protein.

[0027] SEQ ID NO:6 is the predicted amino acid sequence of PcuB encodedby ORF1.5 (SEQ ID NO:102) which has the enzyme activity of PCMH.

[0028] SEQ ID NOs:7-77 are the nucleotide sequences of primers used forsequencing pcu.

[0029] SEQ ID NOs:78-79 are the nucleotide sequences of primers used forcloning a Pseudomonas putida (NCIMB 9869) pchC gene.

[0030] SEQ ID NOs:80-90 are the nucleotide sequences of primers used forsequencing tmoX.

[0031] SEQ ID NO:91 is the nucleotide sequence of the tmoX gene and its5′ non-translated region from Pseudomonas mendocina KR-1.

[0032] SEQ ID NO:92 is the deduced amino acid sequence of TmoX encodedby ORF2.1 (SEQ ID NO:103).

[0033] SEQ ID NOs:93-94 are the nucleotide sequences of primers used forcloning pcu for insertion into pMC3.

[0034] SEQ ID NOs:95-96 are the nucleotide sequence of primers used forconstructing plasmids pPCUR1 and pPCUR2.

[0035] SEQ ID NO:97 is the nucleotide sequence of the primer used to mapthe transcript initiation site of tmoX.

[0036] SEQ ID NO:98 is the nucleotide sequence of the transcriptionalactivator PcuR (ORF1.1).

[0037] SEQ ID NO:99 is the nucleotide sequence of PcuC (ORF1.2).

[0038] SEQ ID NO:100 is the-nucleotide sequence of PcuA (ORF1.3).

[0039] SEQ ID NO:101 is the nucleotide sequence of PcuX (ORF1.4).

[0040] SEQ ID NO:102 is the nucleotide sequence of PcuB (ORF1.5).

[0041] SEQ ID NO:103 is the nucleotide sequence of the tmoX gene fromPseudomonas mendocina KR-1 (ORF2.1).

[0042] SEQ ID NO:104 is a primer used to identify the pobA gene.

[0043] SEQ ID NO:105 is a primer used to identify the pobA gene.

[0044] SEQ ID NO:106 is a primer used to identify the pobA gene.

[0045] SEQ ID NO:107 is a primer used to identify the pobA gene.

[0046] SEQ ID NO:108 is a primer used to identify the pobA gene.

[0047] SEQ ID NO:109 is a primer used to identify the pobA gene.

[0048] SEQ ID NO:110 is a primer used to identify the pobA gene.

[0049] SEQ ID NO:111 is a primer used to identify the pobB gene.

[0050] SEQ ID NO:112 is the nucleotide sequence of the todST genes.

[0051] Applicant has made the following biological deposits under theterms of the Budapest Treaty on the International Recognition of theDeposit of Micro-organisms for the Purposes of Patent Procedure:Depositor Identification International Depository Reference DesignationDate of Deposit Pseudomonas mendocina ATCC 55885 KRC16KDpobA51

[0052] The Depositor has authorized the Applicant to refer to thedeposited material in the application and has given his unreserved andirrevocable consent to the deposited material being made available tothe public in accordance with Rule 28 of the Implementing Regulations tothe European Patent Convention (Rule 28(1)(d) EPC).

DETAILED DESCRIPTION OF THE INVENTION

[0053] PHBA is a valuable monomer for the synthesis of liquidcrystalline polymers (LCP). Applicants have provided methods for thebiological production of PHBA from genetically engineered Pseudomonas,Agrobacterium, or related strains transformed with a pcu operon. Theinstant methods provide PHBA without the high energy cost of syntheticproduction and without producing toxic waste streams. Applicants havealso provided a method for the biological production of p-cresol fromgenetically engineered Escherichia or Pseudomonas.

[0054] The following abbreviations and definitions will be used tointerpret the specification and the claims.

[0055] “para-Hydroxybenzoic acid”, “para-hydroxybenzoate”,“p-hydroxybenzoate” or “4-hydroxybenzoic acid” is abbreviated PHBA.

[0056] “para-Hydroxybenzoate hydroxylase” is abbreviated PHBH.

[0057] “Toluene-4-monooxygenase” is abbreviated TMO.

[0058] “para-Cresol methylhydroxylase” is abbreviated PCMH.

[0059] “para-Hydroxybenzaldehyde dehydrogenase” is abbreviated PHBAD.

[0060] “Ethylenediaminetetraacetic acid” is abbreviated EDTA.

[0061] “Isopropyl-β-D-thiogalactopyranoside” is abbreviated IPTG.

[0062] “Shrimp alkaline phosphatase” is abbreviated SAP.

[0063] “Calf intestinal alkaline phosphatase” is abbreviated CIP.

[0064] “Phenazine ethosulfate” is abbreviated PES.

[0065] “2,6-Dichlorophenol-indophenol” is abbreviated DCPIP.

[0066] “SSC” is the abbreviation for 150 mM NaCl, 15 mM sodium citrate,pH 7.0.

[0067] “TE” is the abbreviation for 10 mM Tris-HCl, 1 mM EDTA, pH 8.0.

[0068] The term “amp” refers to ampicillin.

[0069] The term “chl” refers to chloramphenicol.

[0070] The term “kan” refers to kanamycin.

[0071] The term “strep” refers to streptomycin.

[0072] The term “Pip” refers to peperacillan.

[0073] The term “tet” refers to tetracycline.

[0074] The term “strR” refers to a gene conferring resistance tostreptomycin.

[0075] The terms “TMO degradative pathway” or “TMO enzymatic pathway”refer to the enzymes and genes encoding the enzymes found in somePseudomonas bacteria that are responsible for the degradation oftoluene, p-cresol and similar aromatic substrates. The TMO pathway isoutlined in FIG. 1 and contains at least toluene-4-monooxygenase (TMO),p-cresol methylhydroxylase (PCMH), p-hydroxybenzoaldehyde dehydrogenase(PHBAD), and p-hydroxybenzoate hydroxylase (PHBH).

[0076] The term “aromatic organic substrate” refers to an aromaticcompound that is degraded by the TMO enzymatic pathway. Typical examplesof suitable aromatic substrates are toluene, p-cresol, p-hydroxybenzyl,and p-hydroxybenzaldehyde.

[0077] The terms “plasmid”, “vector” and “cassette” refer to an extrachromosomal element often carrying genes which are not part of thecentral metabolism of the cell, and usually in the form of circulardouble-stranded DNA molecules. Such elements may be autonomouslyreplicating sequences, genome-integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell. “Transformation vector” refers to a specific vectorcontaining a foreign gene and having elements in addition to the foreigngene that facilitate transformation of a particular host cell.

[0078] An “isolated nucleic acid molecule” is a polymer of RNA or DNAthat is single- or double-stranded, optionally containing synthetic,non-natural or altered nucleotide bases. An isolated nucleic acidmolecule in the form of a polymer of DNA may be comprised of one or moresegments of cDNA, genomic DNA or synthetic DNA.

[0079] “Substantially similar” refers to nucleic acid molecules whereinchanges in one or more nucleotide bases result in substitution of one ormore amino acids, but do not affect the functional properties of theprotein encoded by the DNA sequence. “Substantially similar” also refersto nucleic acid molecules wherein changes in one or more nucleotidebases do not affect the ability of the nucleic acid molecule to mediatealteration of gene expression by antisense or co-suppression technology.“Substantially similar” also refers to modifications of the nucleic acidmolecules of the instant invention (such as deletion or insertion of oneor more nucleotide bases) that do not substantially affect thefunctional properties of the resulting transcript vis-a-vis the abilityto mediate alteration of gene expression by antisense or co-suppressiontechnology or alteration of the functional properties of the resultingprotein molecule. The invention encompasses more than the specificexemplary sequences.

[0080] For example, it is well known in the art that alterations in agene which result in the production of a chemically equivalent aminoacid at a given site, but do not effect the functional properties of theencoded protein are common. For the purposes of the present inventionsubstitutions are defined as exchanges within one of the following fivegroups:

[0081] 1. Small aliphatic, nonpolar or slightly polar residues: Ala,Ser, Thr (Pro, Gly);

[0082] 2. Polar, negatively charged residues and their amides: Asp, Asn,Glu, Gln;

[0083] 3. Polar, positively charged residues: His, Arg, Lys;

[0084] 4. Large aliphatic, nonpolar residues: Met, Leu, Ile, Val (Cys);and

[0085] 5. Large aromatic residues: Phe, Tyr, Trp.

[0086] Thus, a codon for the amino acid alanine, a hydrophobic aminoacid, may be substituted by a codon encoding another less hydrophobicresidue (such as glycine) or a more hydrophobic residue (such as valine,leucine, or isoleucine). Similarly, changes which result in substitutionof one negatively charged residue for another (such as aspartic acid forglutamic acid) or one positively charged residue for another (such aslysine for arginine) can also be expected to produce a functionallyequivalent product.

[0087] In many cases, nucleotide changes which result in alteration ofthe N-terminal and C-terminal portions of the protein molecule wouldalso not be expected to alter the activity of the protein.

[0088] Each of the proposed modifications is well within the routineskill in the art, as is determination of retention of biologicalactivity of the encoded products. Moreover, the skilled artisanrecognizes that substantially similar sequences encompassed by thisinvention are also defined by their ability to hybridize, understringent conditions (0.1× SSC, 0.1% SDS, 65° C. and washed with 2× SSC,0.1% SDS followed by 0.1× SSC, 0.1% SDS), with the sequences exemplifiedherein. Preferred substantially similar nucleic acid fragments of theinstant invention are those nucleic acid fragments whose DNA sequencesare at least 80% identical to the DNA sequence of the nucleic acidfragments reported herein. More preferred nucleic acid fragments are atleast 90% identical to the DNA sequence of the nucleic acid fragmentsreported herein. Most preferred are nucleic acid fragments that are atleast 95% identical to the DNA sequence of the nucleic acid fragmentsreported herein.

[0089] A nucleic acid fragment is “hybridizable” to another nucleic acidfragment, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid fragment can anneal to the other nucleic acidfragment under the appropriate conditions of temperature and solutionionic strength. Hybridization and washing conditions are well known andexemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. MolecularCloning: A Laboratory Manual, Second Edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor (1989), particularly Chapter 11 andTable 11.1 therein (entirely incorporated herein by reference). Theconditions of temperature and ionic strength determine the “stringency”of the hybridization. For preliminary screening for homologous nucleicacids, low stringency hybridization conditions, corresponding to a Tm of55°, can be used, e.g., 5× SSC, 0.1% SDS, 0.25% milk, and no formamide;or 30% formamide, 5× SSC, 0.5% SDS. Moderate stringency hybridizationconditions correspond to a higher Tm, e.g., 40% formamide, with 5× or 6×SSC. Hybridization requires that the two nucleic acids containcomplementary sequences, although depending on the stringency of thehybridization, mismatches between bases are possible. The appropriatestringency for hybridizing nucleic acids depends on the length of thenucleic acids and the degree of complementation, variables well known inthe art. The greater the degree of similarity or homology between twonucleotide sequences, the greater the value of Tm for hybrids of nucleicacids having those sequences. The relative stability (corresponding tohigher Tm) of nucleic acid hybridization decreases in the followingorder: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100nucleotides in length, equations for calculating Tm have been derived(see Sambrook et al., supra, 9.50-9.51). For hybridization with shorternucleic acids, i.e., oligonucleotides, the position of mismatchesbecomes more important, and the length of the oligonucleotide determinesits specificity (see Sambrook et al., supra, 11.7-11.8). In oneembodiment the length for a hybridizable nucleic acid is at least about10 nucleotides. Preferable a minimum length for a hybridizable nucleicacid is at least about 15 nucleotides; more preferably at least about 20nucleotides; and most preferably the length is at least 30 nucleotides.Furthermore, the skilled artisan will recognize that the temperature andwash solution salt concentration may be adjusted as necessary accordingto factors such as length of the probe.

[0090] A “substantial portion” refers to an amino acid or nucleotidesequence which comprises enough of the amino acid sequence of apolypeptide or the nucleotide sequence of a gene to afford putativeidentification of that polypeptide or gene, either by manual evaluationof the sequence by one skilled in the art, or by computer-automatedsequence comparison and identification using algorithms such as BLAST(Basic Local Alignment Search Tool; Altschul et al., J. Mol. Biol.215:403-410 (1993); see also www.ncbi.nlm.nih.gov/BLAST/). In general, asequence of ten or more contiguous amino acids or thirty or morenucleotides is necessary in order to putatively identify a polypeptideor nucleic acid sequence as homologous to a known protein or gene.Moreover, with respect to nucleotide sequences, gene specificoligonucleotide probes comprising 20-30 contiguous nucleotides may beused in sequence-dependent methods of gene identification (e.g.,Southern hybridization) and isolation (e.g., in situ hybridization ofbacterial colonies or bacteriophage plaques). In addition, shortoligonucleotides of 12-15 bases may be used as amplification primers inPCR in order to obtain a particular nucleic acid molecule comprising theprimers. Accordingly, a “substantial portion” of a nucleotide sequencecomprises enough of the sequence to afford specific identificationand/or isolation of a nucleic acid molecule comprising the sequence. Theinstant specification teaches partial or complete amino acid andnucleotide sequences encoding one or more particular plant proteins. Theskilled artisan, having the benefit of the sequences as reported herein,may now use all or a substantial portion of the disclosed sequences forthe purpose known to those skilled in the art. Accordingly, the instantinvention comprises the complete sequences as reported in theaccompanying Sequence Listing, as well as substantial portions of thosesequences as defined above.

[0091] The term “complementary” describes the relationship betweennucleotide bases that are capable to hybridizing to one another. Forexample, with respect to DNA, adenosine is complementary to thymine andcytosine is complementary to guanine. Accordingly, the instant inventionalso includes isolated nucleic acid molecules that are complementary tothe complete sequences as reported in the accompanying Sequence Listingas well as those substantially similar nucleic acid sequences.

[0092] The term “percent identity”, as known in the art, is arelationship between two or more polypeptide sequences or two or morepolynucleotide sequences, as determined by comparing the sequences. Inthe art, “identity” also means the degree of sequence relatednessbetween polypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”and “similarity” can be readily calculated by known methods, includingbut not limited to those described in: Computational Molecular Biology;Lesk, A. M., Ed.; Oxford University Press: New York, 1988; Biocomputing:Informatics and Genome Projects; Smith, D. W., Ed.; Academic Press: NewYork, 1993; Computer Analysis of Sequence Data, Part I; Griffin, A. M.and Griffin, H. G., Eds.; Humana Press: New Jersey, 1994; SequenceAnalysis in Molecular Biology; von Heinje, G., Ed.; Academic Press: NewYork, 1987; and Sequence Analysis Primer; Gribskov, M. and Devereux, J.,Eds.; Stockton Press: New York, 1991. Preferred methods to determineidentity are designed to give the largest match between the sequencestested.

[0093] Methods to determine identity and similarity are codified inpublicly available computer programs. Preferred computer program methodsto determine identity and similarity between two sequences include, butare not limited to, the GCG Pileup program found in the GCG programpackage, using the Needleman and Wunsch algorithm with their standarddefault values of gap creation penalty=12 and gap extension penalty=4(Devereux et al., Nucleic Acids Res. 12:387-395 (1984)), BLASTP, BLASTN,and FASTA (Pearson et al., Proc. Natl. Acad. Sci. USA 85:2444-2448(1988). The BLASTX program is publicly available from NCBI and othersources (BLAST Manual, Altschul et al., Natl. Cent. Biotechnol. Inf.,Natl. Library Med. (NCBI NLM) NIH, Bethesda, Md. 20894; Altschul et al.,J. Mol. Biol. 215:403-410 (1990); Altschul et al., “Gapped BLAST andPSI-BLAST: a new generation of protein database search programs”,Nucleic Acids Res. 25:3389-3402 (1997)). Another preferred method todetermine percent identity, is by the method of DNASTAR proteinalignment protocol using the Jotun-Hein algorithm (Hein et al., MethodsEnzymol. 183:626-645 (1990)). Default parameters for the Jotun-Heinmethod for alignments are: for multiple alignments, gap penalty=11, gaplength penalty=3; for pairwise alignments ktuple=6. As an illustration,by a polynucleotide having a nucleotide sequence having at least, forexample, 95% “identity” to a reference nucleotide sequence it isintended that the nucleotide sequence of the polynucleotide is identicalto the reference sequence except that the polynucleotide sequence mayinclude up to five point mutations per each 100 nucleotides of thereference nucleotide sequence. In other words, to obtain apolynucleotide having a nucleotide sequence at least 95% identical to areference nucleotide sequence, up to 5% of the nucleotides in thereference sequence may be deleted or substituted with anothernucleotide, or a number of nucleotides up to 5% of the total nucleotidesin the reference sequence may be inserted into the reference sequence.These mutations of the reference sequence may occur at the 5′ or 3′terminal positions of the reference nucleotide sequence or anywherebetween those terminal positions, interspersed either individually amongnucleotides in the reference sequence or in one or more contiguousgroups within the reference sequence. Analogously, by a polypeptidehaving an amino acid sequence having at least, for example, 95% identityto a reference amino acid sequence is intended that the amino acidsequence of the polypeptide is identical to the reference sequenceexcept that the polypeptide sequence may include up to five amino acidalterations per each 100 amino acids of the reference amino acid. Inother words, to obtain a polypeptide having an amino acid sequence atleast 95% identical to a reference amino acid sequence, up to 5% of theamino acid residues in the reference sequence may be deleted orsubstituted with another amino acid, or a number of amino acids up to 5%of the total amino acid residues in the reference sequence may beinserted into the reference sequence. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

[0094] The term “percent homology” refers to the extent of amino acidsequence identity between polypeptides. When a first amino acid sequenceis identical to a second amino acid sequence, then the first and secondamino acid sequences exhibit 100% homology. The homology between any twopolypeptides is a direct function of the total number of matching aminoacids at a given position in either sequence, e.g., if half of the totalnumber of amino acids in either of the two sequences are the same thenthe two sequences are said to exhibit 50% homology.

[0095] “Codon degeneracy” refers to divergence in the genetic codepermitting variation of the nucleotide sequence without effecting theamino acid sequence of an encoded polypeptide. Accordingly, the instantinvention relates to any nucleic acid molecule that encodes all or asubstantial portion of the amino acid sequence encoding the PcuR, PcuC,PcuA, PcuX and PcuB proteins as set forth in SEQ ID NO:2 through SEQ IDNO:6, and also to any nucleic acid molecule that encodes all or asubstantial portion of the amino acid sequence encoding the TmoX proteinas set forth in SEQ ID NO:92. The skilled artisan is well aware of the“codon-bias” exhibited by a specific host cell in usage of nucleotidecodons to specify a given amino acid. Therefore, when synthesizing agene for improved expression in a host cell, it is desirable to designthe gene such that its frequency of codon usage approaches the frequencyof preferred codon usage of the host cell.

[0096] “Synthetic genes” can be assembled from oligonucleotide buildingblocks that are chemically synthesized using procedures known to thoseskilled in the art. These building blocks are ligated and annealed toform gene segments which are then enzymatically assembled to constructthe entire gene. “Chemically synthesized”, as related to a sequence ofDNA, means that the component nucleotides were assembled in vitro.Manual chemical synthesis of DNA may be accomplished using wellestablished procedures, or automated chemical synthesis can be performedusing one of a number of commercially available machines. Accordingly,the genes can be tailored for optimal gene expression based onoptimization of nucleotide sequence to reflect the codon bias of thehost cell. The skilled artisan appreciates the likelihood of successfulgene expression if codon usage is biased towards those codons favored bythe host. Determination of preferred codons can be based on a survey ofgenes derived from the host cell where sequence information isavailable.

[0097] “Gene” refers to a nucleic acid molecule that expresses aspecific protein, including regulatory sequences preceding (5′non-coding sequences) and following (3′ non-coding sequences) the codingsequence. “Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers to any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

[0098] “Coding sequence” refers to a DNA sequence that codes for aspecific amino acid sequence.

[0099] “Suitable regulatory sequences” refer to nucleotide sequenceslocated upstream (5′ non-coding sequences), within, or downstream (3′non-coding sequences) of a coding sequence, and which influence thetranscription, RNA processing or stability, or translation of theassociated coding sequence. Regulatory sequences may include promoters,translation leader sequences, introns, and polyadenylation recognitionsequences.

[0100] “Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. An “enhancer” is a DNA sequencewhich can stimulate promoter activity and may be an innate element ofthe promoter or a heterologous element inserted to enhance the level ortissue-specificity of a promoter. Promoters may be derived in theirentirety from a native gene, or be composed of different elementsderived from different promoters found in nature, or even comprisesynthetic DNA segments. It is understood by those skilled in the artthat different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental conditions. Promoters whichcause a gene to be expressed in most cell types at most times arecommonly referred to as “constitutive promoters”. It is furtherrecognized that since in most cases the exact boundaries of regulatorysequences have not been completely defined, DNA molecules of differentlengths may have identical promoter activity.

[0101] The “translation leader sequence” refers to a DNA sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner et al., Mol. Biotech. 3:225(1995)).

[0102] The “3′ non-coding sequences” refer to DNA sequences locateddownstream of a coding sequence and include recognition sequences andother sequences encoding regulatory signals capable of affecting mRNAprocessing or gene expression. The use of different 3 non-codingsequences is exemplified by Ingelbrecht et al., Plant Cell 1:671-680(1989).

[0103] “RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from post-transcriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated into proteinby the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to and derived from mRNA. “Sense” RNA refers to RNAtranscript that includes the mRNA and so can be translated into proteinby the cell. “Antisense RNA” refers to a RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene (U.S. Pat. No. 5,107,065).The complementarity of an antisense RNA may be with any part of thespecific gene transcript, i.e., at the 5′ non-coding sequence, 3′non-coding sequence, introns, or the coding sequence. “Functional RNA”refers to antisense RNA, ribozyme RNA, or other RNA that is nottranslated yet and has an effect on cellular processes.

[0104] The term “operably linked” refers to the association of nucleicacid sequences on a single nucleic acid molecule so that the function ofone is affected by the other. For example, a promoter is operably linkedwith a coding sequence when it affects the expression of that codingsequence (i.e., that the coding sequence is under the transcriptionalcontrol of the promoter). Coding sequences can be operably linked toregulatory sequences in sense or antisense orientation.

[0105] The term “expression”, as used herein, refers to thetranscription and stable accumulation of sense (mRNA) or antisense RNAderived from the nucleic acid molecule of the invention. Expression mayalso refer to translation of mRNA into a polypeptide. “Antisenseinhibition” refers to the production of antisense RNA transcriptscapable of suppressing the expression of the target protein.“Overexpression” refers to the production of a gene product intransgenic organisms that exceeds levels of production in normal ornon-transformed organisms. “Co-suppression” refers to the production ofsense RNA transcripts capable of suppressing the expression of identicalor substantially similar foreign or endogenous genes (U.S. Pat. No.5,231,020).

[0106] “Altered levels” refers to the production of gene product(s) inorganisms in amounts or proportions that are not characteristic ofnormal, wild-type, or non-transformed organisms. The altered level maybe either an increase or decrease in the amount or proporiton of geneproduct relative to that produced by the normal, wild-type, ornon-transformed organism.

[0107] “Mature” protein refers to a post-translationally processedpolypeptide; i.e., one from which any pre- or propeptides present in theprimary translation product have been removed. “Precursor” proteinrefers to the primary product of translation of mRNA; i.e., with pre-and propeptides still present. Pre- and propeptides may be but are notlimited to intracellular localization signals.

[0108] A “fragment” constitutes a fraction of the DNA sequence of theparticular region.

[0109] “Transformation” refers to the transfer of a nucleic acidfragment into the genome of a host organism, resulting in geneticallystable inheritance. Host organisms containing the transformed nucleicacid fragments are referred to as “transgenic” or “recombinant” or“transformed” organisms.

[0110] The terms “plasmid”, “vector” and “cassette” refer to an extrachromosomal element often carrying genes which are not part of thecentral metabolism of the cell, and usually in the form of circulardouble-stranded DNA fragments. Such elements may be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell. “Transformation cassette” refers to a specific vectorcontaining a foreign gene and having elements in addition to the foreigngene that facilitate transformation of a particular host cell.“Expression cassette” refers to a specific vector containing a foreigngene and having elements in addition to the foreign gene that allow forenhanced expression of that gene in a foreign-host.

[0111] The term “expression” refers to the transcription and translationto gene product from a gene coding for the sequence of the gene product.In the expression, a DNA chain coding for the sequence of gene productis first transcribed to a complimentary RNA which is often a messengerRNA and, then, the transcribed messenger RNA is translated into theabove-mentioned gene product if the gene product is a protein.

[0112] The terms “restriction endonuclease” and “restriction enzyme”refer to an enzyme which binds and cuts within a specific nucleotidesequence within double-stranded DNA.

[0113] “Polymerase Chain Reaction” and “PCR” refer to a method thatresults in the linear or logarithmic amplification of nucleic acidmolecules. PCR generally requires a replication composition consistingof, for example, nucleotide triphosphates, two primers with appropriatesequences, DNA or RNA polymerase and proteins. These reagents anddetails describing procedures for their use in amplifying nucleic acidsare provided in U.S. Pat. No. 4,683,202 (1987, Mullis et al.) and U.S.Pat. No. 4,683,195 (1986, Mullis et al.).

[0114] The term “sequence analysis software” refers to any computeralgorithm or software program that is useful for the analysis ofnucleotide or amino acid sequences. “Sequence analysis software” may becommercially available or independently developed. Typical sequenceanalysis software will include but is not limited to the GCG suite ofprograms (Wisconsin Package Version 9.0, Genetics Computer Group (GCG),Madison, Wis.), BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol.215:403-410 (1990), and DNASTAR (DNASTAR, Inc. 1228 S. Park St. Madison,Wis. 53715 USA), and the FASTA program incorporating the Smith-Watermanalgorithm (W. R. Pearson, Comput. Methods Genome Res., [Proc. Int.Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor.Publisher: Plenum, New York, N.Y.). Within the context of thisapplication it will be understood that where sequence analysis softwareis used for analysis, that the results of the analysis will be based onthe “default values” of the program referenced, unless otherwisespecified. As used herein “default values” will mean any set of valuesor parameters which originally load with the software when firstinitialized.

[0115] The term “carbon source” refers to a substrate suitable forbacterial cell growth that is distinct from the aromatic substrate.Suitable carbon substrates include but are not limited to glucose,succinate, lactate, acetate, ethanol, monosaccharides, oligosaccharides,polysaccharides, or mixtures thereof.

[0116] The term “suicide vector” refers to a vector generally containinga foreign DNA fragment to be expressed in a suitable host cell, coupledwith a genetic element that will be lethal to the host cell unless thecell is able to express the foreign DNA. “Suicide vector” is alsounderstood to mean a non-replicating vector capable of transfecting ahost cell and facilitating the incorporation of foreign DNA into thegenome of the host cell. Such a vector does not replicate and is thusdestroyed after incorporation of the heterologous DNA. Examples ofcommon suicide vectors and their construction may be found in Sambrook,J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A LaboratoryManual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y.,1989.

[0117] The meaning of abbreviations is as follows: “h” means hour(s),“min” means minute(s), “sec” means second(s), “d” means day(s), “L”means microliteres, “mL” means milliliters and “L” means liters.

[0118] The nucleic acid fragments of the instant invention may be usedto isolate genes encoding homologous proteins from the same or othermicrobial species. Isolation of homologous genes usingsequence-dependent protocols is well known in the art. Examples ofsequence-dependent protocols include, but are not limited to, methods ofnucleic acid hybridization, and methods of DNA and RNA amplification asexemplified by various uses of nucleic acid amplification technologies(e.g. polymerase chain reaction (PCR), Mullis et al., U.S. Pat. No.4,683,202), ligase chain reaction (LCR), Tabor, S. et al., Proc. Acad.Sci. USA 82, 1074, (1985)) or strand displacement amplification (SDA,Walker, et al., Proc. Natl. Acad. Sci. U.S.A., 89, 392, (1992)).

[0119] For example, genes encoding similar proteins or polypetides tothose of the instant invention could be isolated directly by using allor a portion of the instant nucleic acid fragments as DNA hybridizationprobes to screen libraries from any desired bacteria using methodologywell known to those skilled in the art. Specific oligonucleotide probesbased upon the instant nucleic acid sequences can be designed andsynthesized by methods known in the art (Maniatis). Moreover, the entiresequences can be used directly to synthesize DNA probes by methods knownto the skilled artisan such as random primers DNA labeling, nicktranslation, or end-labeling techniques, or RNA probes using availablein vitro transcription systems. In addition, specific primers can bedesigned and used to amplify a part of or full-length of the instantsequences. The resulting amplification products can be labeled directlyduring amplification reactions or labeled after amplification reactions,and used as probes to isolate full length DNA fragments under conditionsof appropriate stringency.

[0120] Typically, in PCR-type amplification techniques, the primers havedifferent sequences and are not complementary to each other. Dependingon the desired test conditions, the sequences of the primers should bedesigned to provide for both efficient and faithful replication of thetarget nucleic acid. Methods of PCR primer design are common and wellknown in the art. (Thein and Wallace, “The use of oligonucleotide asspecific hybridization probes in the Diagnosis of Genetic Disorders”, inHuman Genetic Diseases: A Practical Approach, K. E. Davis Ed., (1986)pp. 33-50 IRL Press, Herndon, Va.); Rychlik, W. (1993) In White, B. A.(ed.), Methods in Molecular Biology, Vol.15, pages 31-39, PCR Protocols:Current Methods and Applications. Humania Press, Inc., Totowa, N.J.).

[0121] Generally two short segments of the instant sequences may be usedin polymerase chain reaction protocols to amplify longer nucleic acidfragments encoding homologous genes from DNA or RNA. The polymerasechain reaction may also be performed on a library of cloned nucleic acidfragments wherein the sequence of one primer is derived from the instantnucleic acid fragments, and the sequence of the other primer takesadvantage of the presence of the polyadenylic acid tracts to the 3′ endof the mRNA precursor encoding microbial genes.

[0122] Alternatively, the second primer sequence may be based uponsequences derived from the cloning vector. For example, the skilledartisan can follow the RACE protocol (Frohman et al., PNAS USA 85:8998(1988)) to generate cDNAs by using PCR to amplify copies of the regionbetween a single point in the transcript and the 3′ or 5′ end. Primersoriented in the 3′ and 5′ directions can be designed from the instantsequences. Using commercially available 3′ RACE or 5′ RACE systems(BRL), specific 3′ or 5′ cDNA fragments can be isolated (Ohara et al.,PNAS USA 86:5673 (1989); Loh et al., Science 243:217 (1989)).

[0123] Alternatively the instant sequences may be used as hybridizationreagents for the identification of homologs. The basic components of anucleic acid hybridization test include a probe, a sample suspected ofcontaining the gene or gene fragment of interest, and a specifichybridization method. Probes of the present invention are typicallysingle stranded nucleic acid sequences which are complementary to thenucleic acid sequences to be detected. Probes are “hybridizable” to thenucleic acid sequence to be detected. The probe length can vary from 5bases to tens of thousands of bases, and will depend upon the specifictest to be done. Typically a probe length of about 15 bases to about 30bases is suitable. Only part of the probe molecule need be complementaryto the nucleic acid sequence to be detected. In addition, thecomplementarity between the probe and the target sequence need not beperfect. Hybridization does occur between imperfectly complementarymolecules with the result that a certain fraction of the bases in thehybridized region are not paired with the proper complementary base.

[0124] Hybridization methods are well defined. Typically the probe andsample must be mixed under conditions which will permit nucleic acidhybridization. This involves contacting the probe and sample in thepresence of an inorganic or organic salt under the proper concentrationand temperature conditions. The probe and sample nucleic acids must bein contact for a long enough time that any possible hybridizationbetween the probe and sample nucleic acid may occur. The concentrationof probe or target in the mixture will determine the time necessary forhybridization to occur. The higher the probe or target concentration theshorter the hybridization incubation time needed. Optionally achaotropic agent may be added. The chaotropic agent stabilizes nucleicacids by inhibiting nuclease activity. Furthermore, the chaotropic agentallows sensitive and stringent hybridization of short oligonucleotideprobes at room temperature [Van Ness and Chen (1991) Nucl. Acids Res.19:5143-5151]. Suitable chaotropic agents include guanidinium chloride,guanidinium thiocyanate, sodium thiocyanate, lithium tetrachloroacetate,sodium perchlorate, rubidium tetrachloroacetate, potassium iodide, andcesium trifluoroacetate, among others. Typically, the chaotropic agentwill be present at a final concentration of about 3M. If desired, onecan add formamide to the hybridization mixture, typically 30-50% (v/v).

[0125] Various hybridization solutions can be employed. Typically, thesecomprise from about 20 to ⁶⁰% volume, preferably 30%, of a polar organicsolvent. A common hybridization solution employs about 30-50% v/vformamide, about 0.15 to 1M sodium chloride, about 0.05 to 0.1M buffers,such as sodium citrate, Tris-HCl, PIPES or HEPES (pH range about 6-9),about 0.05 to 0.2% detergent, such as sodium dodecylsulfate, or between0.5-20 mM EDTA, FICOLL (Pharmacia Inc.) (about 300-500 kilodaltons),polyvinylpyrrolidone (about 250-500 kdal), and serum albumin. Alsoincluded in the typical hybridization solution will be unlabeled carriernucleic acids from about 0.1 to 5 mg/mL, fragmented nucleic DNA, e.g.,calf thymus or salmon sperm DNA, or yeast RNA, and optionally from about0.5 to 2% wt./vol. glycine. Other additives may also be included, suchas volume exclusion agents which include a variety of polarwater-soluble or swellable agents, such as polyethylene glycol, anionicpolymers such as polyacrylate or polymethylacrylate, and anionicsaccharidic polymers, such as dextran sulfate.

[0126] Nucleic acid hybridization is adaptable to a variety of assayformats. One of the most suitable is the sandwich assay format. Thesandwich assay is particularly adaptable to hybridization undernon-denaturing conditions. A primary component of a sandwich-type assayis a solid support. The solid support has adsorbed to it or covalentlycoupled to it immobilized nucleic acid probe that is unlabeled andcomplementary to one portion of the sequence.

[0127] Availability of the instant nucleotide and deduced amino acidsequences facilitates immunological screening DNA expression libraries.Synthetic peptides representing portions of the instant amino acidsequences may be synthesized. These peptides can be used to immunizeanimals to produce polyclonal or monoclonal antibodies with specificityfor peptides or proteins comprising the amino acid sequences. Theseantibodies can be then be used to screen DNA expression libraries toisolate full-length DNA clones of interest (Lerner, R. A. Adv. Immunol.36:1 (1984); Maniatis).

[0128] The genes and gene products of the instant sequences may beproduced in heterologous host cells, particularly in the cells ofmicrobial hosts. Expression in recombinant microbial hosts may be usefulfor the expression of various pathway intermediates; for the modulationof pathways already existing in the host for the synthesis of newproducts heretofore not possible using the host. Additionally the geneproducts may be useful for conferring higher growth yields of the hostor for enabling alternative growth mode to be utilized.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0129] TMO-Containing Bacterial Strains:

[0130] Bacterial cells preferred in the present invention are those thatpossesses the TMO pathway. Such strains are generally restricted to thegenus Pseudomonas and include, but are not limited to, Pseudomonasputida and Pseudomonas mendocina. Strains of Burkholderia andAcinetobacter are also suitable as host cells.

[0131] Strains of Pseudomonas containing the TMO pathway are known tooxidize toluene to form intermediates of the tricarboxylic acid cycle.PHBA as well as other intermediates, such as p-cresol, p-hydroxybenzylalcohol and p-hydroxybenzadehyde, are formed in the upper pathway, whichmetabolizes toluene to the ring cleavage substrate (FIG. 1). In wildtypePseudomonas strains, PHBA is immediately converted to protocatechuate(PCA) as it is formed. The biochemistry of the enzymes involved in theupper pathway have been described for several Pseudomonas strains(Romine et al., supra).

[0132] Batch and Continuous Fermentations:

[0133] The present process uses a batch method of fermentation. Aclassical batch fermentation is a closed system where the composition ofthe medium is set at the beginning of the fermentation and not subjectedto artificial alterations during the fermentation. Thus, at thebeginning of the fermentation the medium is inoculated with the desiredorganism or organisms and fermentation is permitted to occur addingnothing to the system. Typically, however, a batch fermentation is“batch” with respect to the addition of carbon source and attempts areoften made at controlling factors such as pH and oxygen concentration.In batch systems the metabolite and biomass compositions of the systemchange constantly up to the time the fermentation is stopped. Withinbatch cultures, cells moderate through a static lag phase to a highgrowth log phase and finally to a stationary phase where growth rate isdiminished or halted. If untreated, cells in the stationary phase willeventually die.

[0134] A variation on the standard batch system is the fed-batch system.Fed-batch fermentation processes are also suitable in the presentinvention and comprise a typical batch system with the exception thatthe substrate is added in increments as the fermentation progresses.Fed-batch systems are useful when catabolite repression is apt toinhibit the metabolism of the cells and where it is desirable to havelimited amounts of substrate in the medium. An advantage of thefed-batch system is that it is more amenable to the use of toxic orimmiscible aromatic substrates such as toluene or p-cresol. Using afed-batch system it is possible to maintain a steady concentration ofsubstrate at non-toxic levels while accommodating maximum bioconversionof the substrate to product.

[0135] The production of PHBA from aromatic compounds such as toluene orp-cresol will be limited by the amount of the aromatic substrate andcarbon sources added. In simple batch fermentation, production will belimited by the amount of toluene initially added. Since toluene is toxicand has limited solubility in water, its low initial concentration willgovern the amount of PHBA produced. The ability to run the process atsuch a low toluene (i.e., 30-60 ppm) allows operation below a lowerexplosive limit which for toluene is 120 ppm. This low limit is a clearsafety advantage to the process. Fed-batch techniques where the carbonsource and toluene are added at rates which are similar to theutilization of these compounds will keep the toluene concentration inthe medium low and can significantly increase the amount of PHBAproduced.

[0136] Batch and fed-batch fermentations are common and well known inthe art and examples may be found in, for example Brock, Thomas D. InBiotechnology: A Textbook of Industrial Microbiology, 2nd ed.; SinauerAssociates, Inc.: Sunderland, Mass., 1989 or Deshpande, Mukund V. Appl.Biochem. Biotechnol. 36:227 (1992).

[0137] Although the present invention is performed in batch mode, it iscontemplated that the method would be adaptable to continuousfermentation methods. Continuous fermentation is an open system where adefined fermentation medium is added continuously to a bioreactor and anequal amount of conditioned medium is removed simultaneously forprocessing. Continuous fermentation generally maintains the cultures ata constant high density where cells are primarily in log phase growth.

[0138] Continuous fermentation allows for the modulation of one factoror any number of factors that affect cell growth or end productconcentration. For example, one method will maintain a limiting nutrientsuch as the carbon source or nitrogen source at low concentration andallow all other parameters to be in excess. In other systems a number offactors affecting growth can be altered continuously while the cellconcentration, measured by medium turbidity, is kept constant.Continuous systems strive to maintain steady state growth conditions andthus the cell loss due to medium being drawn off must be balancedagainst the cell growth rate in the fermentation. Methods of modulatingnutrients and growth factors for continuous fermentation processes aswell as techniques for maximizing the rate of product-formation are wellknown in the art of industrial microbiology and a variety of methods aredetailed by Brock, supra.

[0139] It is contemplated that the present invention may be practicedusing either batch, fed-batch or continuous processes and that any knownmode of fermentation would be suitable. Additionally, it is contemplatedthat cells may be immobilized on a substrate as whole cell catalysts andsubjected to fermentation conditions for PHBA production.

[0140] Carbon Source:

[0141] A variety of carbon sources are suitable in the present inventionand include but are not limited to materials (such as succinate,lactate, acetate, ethanol), monosaccharides (such as glucose andfructose), oligosaccharides (such as lactose or sucrose),polysaccharides (such as starch or cellulose), or mixtures thereof andunpurified mixtures from renewable feedstocks (such as cheese wheypermeate, cornsteep liquor, sugar beet molasses, and barley malt). Theneeds of the desired production cell dictate the choice of the carbonsubstrate. For the purposes of the present invention, glucose ispreferred.

[0142] Aromatic Substrates:

[0143] A variety of aromatic substrates may be used in the presentinvention, including but not limited to toluene, p-cresol,p-hydroxybenzyl alcohol, p-hydroxybenzaldehyde, and any aromaticcompounds where the chemical structure is similar to toluene-and theintermediates of the TMO pathway (i.e., compounds that are subject todegradation by the TMO pathway).

[0144] The concentration of the aromatic substrate (such as toluene andp-cresol) and of the carbon source in the medium are limiting factorsfor the production of PHBA. Preferred concentrations of toluene are fromabout 30 ppm to about 500 ppm where a range of about 30 ppm to about 60ppm is most preferred. There are tolerant strains that can fermenttoluene at >500 ppm and there are sensitive strains that may operate ata more suitable range of 1-5 ppm. The preferred concentration ofp-cresol for Pseudomonas mendocina is from about 1 mM to about 5 mM.More tolerant strains are expected as well as more sensitive strains.

[0145] The p-cresol concentration needs to be adjusted accordingly.

EXAMPLES

[0146] The present invention is further defined in the followingExamples. It should be understood that these Examples, while indicatingpreferred embodiments of the invention, are given by way of illustrationonly. From the above discussion and these Examples, one skilled in theart can ascertain the essential characteristics of this invention, andwithout departing from the spirit and scope thereof, can make variouschanges and modifications of the invention to adapt it to various usagesand conditions.

[0147] Procedures for the genetic manipulations of cellular genomes arewell known in the art. Techniques suitable for use in the followingexamples may be found in Sambrook, J. In Molecular Cloning: A LaboratoryManual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989.

[0148] Materials and methods suitable for the maintenance and growth ofbacterial cultures are well known in the art. Techniques suitable foruse in the following examples may be found in Manual of Methods forGeneral Bacteriology; Phillipp Gerhardt, R. G. E. Murray, Ralph N.Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. BriggsPhillips, Eds.; American Society for Microbiology: Washington, D.C.,1994 or Brock, Thomas D. In Biotechnology: A Textbook of IndustrialMicrobiology, 2nd ed.; Sinauer Associates, Inc.: Sunderland, Mass.,1989. All reagents and materials used for the growth and maintenance ofbacterial cells were obtained from Aldrich Chemicals (Milwaukee, WT),DIFCO Laboratories (Detroit, Mich.), GIBCO/BRL (Gaithersburg, Md.), orSigma Chemical Company (St. Louis, Mo.) unless otherwise specified.

[0149] Materials and Growth Conditions

[0150] Cell Strains and Plasmids:

[0151] For General Use:

[0152]Escherichia coli DH5α (Clontech, Palo Alto, Calif.), Escherichiacoli DH10B (Gibco BRL, Gaithersburg, Md.), Escherichia coli JM105 (ATCC47016), Escherichia coli Top10F′ (Invitrogen, Carlsbad, Calif. 92008)Escherichia coli XL 1-Blue MR (Stratagene, La Jolla, Calif.) andEscherichia coli XL2 Blue (Stratagene, La Jolla, Calif.).

[0153] Sources of DNA for Cloning:

[0154]Pseudomonas mendocina KR-1 (U.S. Pat. No. 5,171,684; Amgen,Thousand Oaks, Calif.), Pseudomonas mendocina KRC16 KDpobA51 (ATCC55885) (PCT/US98/WO 12072; DuPont, Wilmington, Del.) and Pseudomonasputida (NCIMB 9869).

[0155] For Plasmid Mobilization:

[0156]Escherichia coli S17-1 (ATCC 47055).

[0157] For pcuC::lacZ Expression:

[0158]Escherichia coli MC1061 (CGSC 6649).

[0159] For p-Cresol Production:

[0160]Escherichia coli G1724 (Invitrogen, Carlsbad, Calif.), Escherichiacoli JM105 (ATCC 47016) and Pseudomonas putida (ATCC 29607).

[0161] For PHBA Production:

[0162]Agrobacterium rhizogenes (ATCC 15834), Pseudomonas mendocina KRC16KDpobA51 (ATCC 55885) (PCT/US98/12072; DuPont, Wilmington, Del.) andPseudomonas putida (ATCC 29607).

[0163] For tmo and pcu-Encoded Enzyme Synthesis:

[0164]Pseudomonas putida DOT-T1 (Ramos et al., J. Bact.177(14):3911-3916 (1995)). Pseudomonas putida DOT-T1 C5aAR1 hasmutations that inactivate toluene dioxygenase. Pseudomonas putidaDOT-T1E (CECT 5312).

[0165] “ATCC” refers to the American Type Culture Collectioninternational depository located at 10801 University Boulevard,Manassas, Va. 20110-2209, U.S.A. The designations refer to the accessionnumber of the deposited material.

[0166] “CGSC” refers to the E. coli Genetic Stock Center located at 355Osborn Memorial Laboratories, Department of Biology, Yale University,New Haven, Conn. 06520-8104. The designations refer to the accessionnumber of the deposited material.

[0167] “NCCB” refers to the Netherlands Culture Collection of Bacteria,Utrecht University, P.O. Box 80.056, 3508 TB Utrecht, the Netherlands.The designations refer to the accession number of the depositedmaterial.

[0168] “NCIMB” refers to the National Collection of Industrial andMarine Bacteria Ltd located at 23 St. Machar Drive, Aberdeen, AB2 1RY,U.K. The designations refer to the accession number of the depositedmaterial.

[0169] Growth Conditions:

[0170] Typically, studies were conducted by shaking cultures in 125 mLor 250 μL flasks. Experiments using toluene were conducted in 125 mLsealed flasks. Minimal (lean) medium with glucose as the carbon sourceand ammonia as the nitrogen source was used most extensively. Yeastextract, when added to obtain a “rich” medium, was at 0.5-1.0 g/L. Someof the PHBA production examples included two stages, where the cellswere first grown to a suitable cell density in minimal medium containingglucose, followed by transfer to a production medium containing anaromatic substrate for PHBA production. Culture conditions weremodulated according to the method of growth and optimized for theproduction of PHBA. The pH of the cultures should be maintained within arange of about from 6.3 to 7.9. A range of about 7.2 to 7.7 is mostpreferred. Other media amenable to the procedures of the presentinvention are common in the art and are fully described in Manual ofMethods for General Bacteriology (P. Gerhardt, R. G. E. Murray, R. N.Costilow, E. W. Nester, W. A. Wood, N. R. Krieg and G. B. Phillips,Eds.; American Society for Microbiology: Washington, D.C., 1994).

Example 1 Cloning and Sequencing of the Pseudomonas mendocina pcu Operon

[0171] Preparation of Genomic DNA:

[0172]Pseudomonas mendocina KRC16 KDpobA51 (ATCC 55885) containing anomega-disrupted pobA-1 gene was used as the source of genomic DNA. Thecells of a 50 mL overnight stationary phase culture were collected bycentrifugation at 6,000 rpm, 4° C. for 10 min. The supernatant wasdecanted and the pellets resuspended with 5 mL TEG (25 mM Tris-HCl, 10mM EDTA, 50 mM glucose, pH 8.0). About 1.5 mL of RNAse (100 μg/mL) wasadded into the mixture. The sample was kept at room temperature for 5min, and then extracted twice with an equal volume of phenol. The twophases were separated by a centrifugation at 6,000 rpm for 10 min. Theaqueous phase was extracted twice with phenol:chloroform (1:1). Twovolumes of 100% ethanol were added to the aqueous phase to precipitateDNA. After 20 min the solution was centrifuged at 10,000 rpm, and thepellet was collected, dried, and resuspended in 2 to 5 mL TE buffer. TheDNA sample was dialyzed against TE buffer at 4° C. overnight.

[0173] Construction of a Genomic Library:

[0174] 10 μg of genomic DNA was digested with 100 units of BstYIrestriction endonuclease at 60° C., and samples removed at 2, 5, 10, 20and 30 min intervals in order to obtain partially digested DNA. Thepooled partial digests were treated with phenol:chloroform (1:1),chloroform, and two volumes ethanol added to precipitate the DNA.Resuspended DNA (1.6 μg) was ligated at 4° C. overnight using T4 DNAligase and <1 μg SuperCos 1 (Stratagene, LaJolla, Calif.) that had beendigested with XbaI, dephosphorylated with CIP, and then digested withBamHI. Each enzyme treatment was followed by extraction with equalvolumes of phenol:chloroform (1:1), chloroform, and precipitated with 2volumes of ethanol. Ligated DNA was recovered in bacteriophage lambda byin vitro packaging using a Gigapack II Gold Packaging Extract(Stratagene, La Jolla, Calif.).

[0175] Selection of Clones with a pobA-1 Omega Insert:

[0176]Escherichia coli XL 1-Blue MR cells were infected with thepackaged cosmid library and plated on LB medium containing 50 mg/L ampand 25 mg/L strep, and cultured at 37° C. overnight. As a control, partof the packaged library was plated on LB medium containing 50 mg/L ampto determine total number of cosmid containing cells. About 1% of theamp resistant colonies were also strep resistant, and these representedclones that had acquired the omega-inactivated (strR)pobA-1 gene.

[0177] Restriction and Hybridization Analysis of strR Cosmids:

[0178] Plasmids were isolated from 5 mL cultures of strR clones using analkaline lysis method (Bimboim et al., Nucleic Acids Res. 7(6):1513-1523(1979)). The plasmids were digested with the restriction enzymes HindIIIor ClaI and fragments separated by electrophoresis overnight on a 0.7%agarose gel in TBE buffer (89 mM Tris-borate, 89 mM boric acid, 2 mMEDTA). Cosmids were identified by the presence of a 14 kb HindIIIfragment, or a 12.5 kb ClaI fragment as predicted (Wright et al., Appl.Environ. Microbiol 60(1):235-242 (1994)). DNA was transferred from theagarose gel to a GeneScreen Plus nylon membrane (NEN Life ScienceProducts, Boston, Mass.) using a VacuGene XL system (Pharmacia Biotech,Piscataway, N.J.). Depurination of DNA in the gel with 0.25 M HCl for 7min was followed by denaturation with 1.5 M NaCl+0.5 M NaOH for 7 min,neutralization with 1.0 M Tris-HCl pH 7.5+1.5 M NaCl for 7 min, andtransfer to membrane in 20× SSC for 30 min. The nylon membrane wasremoved, washed in 0.4 M NaOH (1 min), in 0.2 M Tris-HCl pH 7.5+1× SSC(1 min), in 2× SSC (1 min), followed by exposure to ultraviolet lightfor about 2 min to produce nucleic acid crosslinking.

[0179] The membrane was prehybridized for 1 h at 65° C. in ahybridization solution containing 5× SSC, 0.1% (w/v) SDS, 0.5% (w/v)blocking reagent (NEN Life Science Products, Boston, Mass.) and 5% (w/v)Dextran Sulfate. The hybridization probe was a heterologous sequence forthe cytochrome c subunit of PCMH from Pseudomonas putida NCIMB 9869. Thecytochrome c subunit gene (pchC) was cloned from DNA purified fromPseudomonas putida NCIMB 9869 by CsCl-ethidium bromide centrifugation(Pemberton et al., J. Bact. 114(1):424-433 (1973)), and amplified by PCRusing primers (SEQ ID NO:78 and SEQ ID NO:79) based on the publishedsequence (Kim et al., J. Bact. 176(20):6349-6361 (1994)). The 100 μL PCRreaction mixture contained: 0.5 mM dNTPs, reaction buffer (finalconcentration of 10 mM Tris-HCl, pH 8.3, 50 MM KCl, 1.5 mM MgCl₂, and0.01% gelatin), 0.1 mg of Pseudomonas putida genomic DNA, and 1 unit ofTaq DNA polymerase. The DNA sample was denatured at 94° C. for 1 min,and annealed at 50° C. for 2 min. Polymerization was performed at 74° C.for 2 min with an increased extention time of 5 sec per cycle. Thepolymerase chain reaction was accomplished by 25 cycles. The PCR DNAfragment was detected and analyzed by electrophoresis on 1% agarose gelswith 0.5 mg/L ethldium bromide, and cloned into the vector pUC18(Pharmacia Biotech, Piscataway, N.J.).

[0180] For ease of identification, the pchC DNA was labeled with afluorescein nucleotide in a 30 μL reaction mixture containing a randomprimer, reaction buffer, fluorescein nucleotide mix (NEN Life ScienceProducts, Boston, Mass.) and Klenow enzyme at 37° C. for 1 h. Thelabeled probe was then hybridized to the membrane-bound genomic DNA inthe same buffer for 16 h at 65° C.

[0181] After hybridization, the membrane was washed for 15 min in 2×SSC, 0.1% SDS, followed by a second 15 min wash in 0.2× SSC, 0.1% SDS at65° C. The membrane was blocked for 1 h in buffer containing 0.5%blocking reagent and then incubated with antifluorescein-horse radishperoxidase conjugate (1:1000) (NEN Life Science Products, Boston, Mass.)at room temperature for 1 h.

[0182] After the incubation the membranes were washed four times for 5min with 0.1 M Tris-HCl pH 7.5, 0.15 M NaCl, and incubated in achemiluminescence reagent (Renaissance nucleic acid chemiluminescentreagent, NEN Life Science Products, Boston, Mass.) for 1 min at roomtemperature, and then exposed to Reflection autoradiography film (NENLife Science Products, Boston, Mass.). Those clones having both thecorrect restriction pattern with HindIII or ClaI, and which hybridizedto the pchC probe, were selected for sub-cloning and sequencing.

[0183] Subcloning and Sequencing:

[0184] A strR cosmid was digested with HindIII and the ˜14 kb insertisolated from a 0.8% agarose gel using the DNA preparation kit GeneClean(Bio101, Vista, Calif.). The isolated fragment was cloned into theHindIII site of the vector pZErO-1 (Invitrogen, Carlsbad, Calif.),transformed into Escherichia coli Top10F′, and selected on LB mediumcontaining 50 mg/L zeocin. Zeocin-resistant clones were screened bydigestion of plasmid minipreps with HindIII, BamHI, SalI/BamHI,ClaI/SphI, and SphI. A plasmid with a digestion pattern indicating thatthe pcu-encoded enzymes were oriented for transcription by the lacpromoter of pZErO-1 was designated pPCU1, and a plasmid with theopposite orientation was designated pPCU2.

[0185] A 3.5 kb NruI/EcoRI fragment was isolated from pPCU1, and a BamHIadaptor (New England Biolabs, Beverly, Mass.) annealed and ligated to 2μg of fragment in a 20 μL reaction containing 2 mM adaptor at 16° C. for16 h. Following a phenol:chloroform (1:1) extraction and ethanolprecipitation, the DNA was dissolved in 12 μL TE, digested with BamHIfor 5 h, and purified by electrophoresis on a 1% agarose gel andisolated with GeneClean as before. The BamHI/EcoRI fragment was clonedinto the EcoRI/BamHI digested vector pK194 (ATCC 37767) to yield plasmidpPCU3. The complete sequence of the pcu operon is shown in SEQ ID NO:1and the nucleotide sequences for the transcriptional activator PcuR (SEQID NO:98), PHBAD (SEQ ID NO:99), the two subunits of PCMH (SEQ ID NO:100and SEQ ID NO:102), and an unidentified open reading frame (SEQ IDNO:101). Also given are the predicted amino acid sequences for thetranscriptional activator PcuR (SEQ ID NO:2), PHBAD (SEQ ID NO:3), thetwo subunits of PCMH (SEQ ID NO:4 and SEQ ID NO:6), and an unidentifiedopen reading frame (SEQ ID NO:5). The DNA was sequenced with syntheticprimers (SEQ ID NO:7 to SEQ ID NO:77) according to standard methods.

[0186] Identification of the PHBAD and PCMH coding sequences was basedon percent homolgy to the corresponding predicted amino acid sequencesfor these enzymes from Pseudomonas putida NCIMB 9866 and 9869 (Kim etal., supra; Cronin et al., DNA Sequence 10(1):7-17 (1999)).Identification of the PcuR transcriptional activator was based onhomology to the predicted amino acid sequence of the TbuTtranscriptional activator of Ralstonia pickettii (Olsen et al., J.Bacteriol. 176(12):3749-3756 (1994)). Based on the work of Cronin et al.(supra), the unidentified open reading frame (SEQ ID NO:5) may be aninner membrane protein. Their analysis by PSORT for the Pseudomonasputida protein predicts it to be an inner membrane protein, and analysisby TMpred predicts it to have one or two transmembrane helices, with thebulk of the protein lying on the cytoplasmic side in either situation.The arrangement of genes in the pcu operon is illustrated in FIG. 2. Thebest homologies to each ORF, and thus their putative function in the pcuoperon, are listed in Table 1. TABLE 1 SEQ ID SEQ ID % % ORF SimilarityIdentified base Peptide Identity^(a) Similarity^(b) E-value^(c) Citation1.1 gi|1657782  98  2 48% 63% 1e-143 J. Bacteriol. 176 (12), 3749-3756transcriptional activator TbuT (1994) (Ralstonia pickettii) 1.2gb|AAA75634.2|  99  3 75% 83% DNA Seq. 10 (1), 7-17 (1999)p-hydroxybenzaldehyde dehydrogenase (Pseudomonas putida) 1.3gb|AAA80319.2| 100  4 60% 74% 3e-25 DNA Seq. 10 (1), 7-17 (1999)p-cresol methylhydroxylase, cytochrome subunit precursor (Pseudomonasputida) 1.4 gb|AAD29836.1|U96338_3 101  5 46% 61% 5e-36 DNA Seq. 10 (1),7-17 (1999) unknown (Pseudomonas putida) 1.5 gb|AAA80318.2| 102  6 78%88% DNA Seq. 10 (1), 7-17 (1999) p-cresol methylhydroxylase,flavoprotein subunit (Pseudomonas putida) 2.1 emb|CAB43725.1| 103 92 81%87% Gene 232, 69-76 (1999) membrane protein (Pseudomonas putida)

Example 2 Cloning the Pseudomonas mendocina tmo Operon

[0187]Pseudomonas mendocina KR-1 was the source of total genomic DNA,and it was isolated as described before for Pseudomonas mendocinaKRC16KDpobA51(ATCC 55885). Total genomic DNA was digested withSstI+XmaI, separated on a 0.8% low-melting agarose gel, and fragments inthe 5-7 kb size range recovered. The purified DNA was ligated to thevector pUC 18 that had been digested with SstI+XmaI, and the ligated DNAtransformed into Escherichia coli JM105. Clones were selected on LBplates containing amp (100 mg/L) and 10 mM tryptophan. Escherichia coliis able to produce indole from tryptophan using tryptophanase, and thetmo-encoded toluene monooxygenase converts the indole tocis-indole-2,3-dihydrodiol, which then forms indoxyl through thespontaneous elimination of water, and is then oxidized by air to indigo.An indigo-producing colony was isolated and the correctly configuredplasmid identified as pTMO1.

Example 3 Construction of pcu and pcu/tmo Expression Plasmids

[0188] Construction of the pcu Plasmid pPCU12:

[0189] pPCU1 was digested with NruI+ApaI and a 2.4 kb fragment wasisolated by electrophoresis on a 1% agarose gel and purified using aGeneClean kit, then ligated to the SmaI+ApaI digested vector pGadGH(Clontech, Palo Alto, Calif.). The ligation was transformed intocompetent Escherichia coli strain DH5α, and transformants were isolatedon LB+amp (100 mg/L) plates. The correct construct, which was identifiedby the band patterns produced with HindIII+BamHI or BamHI+SalI digests,was named pPCU9.5. Next, a 2.6 kb ApaI fragment was isolated from pPCU1by electrophoresis on a 1% agarose gel followed by purification withGeneClean as before. This fragment was cloned into ApaI-digested pPCU9.5which had also been treated with CIP. Clones containing the insertedfragment were distinguished by digestion with ApaI and detected thepresence of the 2.6 kb fragment. The orientation of the insert wasdetermined by the fragmentation pattern of a BglII digest. The plasmidwith the pattern indicating a complete pcu operon was named pPCU10.

[0190] The ˜5 kb BamHI+HindIII fragment from pPCU10 was isolated asbefore and ligated into the BamHI+HindIII sites in the vector pK184(ATCC 37766). The ligation was transformed into ultracompetent XL2 Bluecells. Transformants were selected using LB+kan (50 mg/L) plates. EcoRIand BglII digests were used to determine the correct construct, whichwas named pPCU11. The 5 kb BamHI+HindIII fragment was isolated frompPCU11 as described above, and the single-stranded ends were convertedto double strands with the Klenow fragment of DNA polymerase I. Thevector pRK310 (Ditta et al., Plasmid 13:149-153 (1985)) was digestedwith HindIII, and the single-stranded ends were also treated with theKlenow fragment of DNA polymerase I and then phosphatased with CIP. Thetwo fragments were ligated together and electroporated into ElectromaxDH10B cells. Colonies with plasmids were selected on LB+tet (12.5 mg/L)plates. EcoRI and SalI digests of plasmids from the colonies were usedto identify a clone of the correct construction, named pPCU12.

[0191] Construction of the pcu Plasmid pPCU18:

[0192] A 7.5 kb MluI+NheI pPCU1 fragment was isolated through agarosegel electroporesis followed by purification with GeneClean. It wasligated into the MluI+NheI sites of plasmid pSL1180 (Pharmacia Biotech,Piscataway, N.J.). The ligation was transformed into competent DH5αcells. Transformants were identified by growth on LB+amp (100 mg/L)plates. SalI digests indicated the correct construct, which was namedpPCU17. Plasmid pPCU17 was digested with BamHI+HindIII, and the 7.5 kbpiece of DNA with the pcu genes was isolated as described earlier. Thefragment was cloned into the BamHI+HindIII sites of the vector pGV1120(Leemans et al., Gene 19:361-364 (1982)). Electrocompetent Pseudomonasputida strain DOT-T1 C5aAR1 cells were electroporated with the ligatedDNA. Cells were selected on LB+strep (50 mg/L) plates at 30° C.overnight. Plasmids were isolated from clones grown on the plates anddigested with EcoRI. The plasmid with the correct digest pattern wasnamed pPCU18.

[0193] Construction of pcuC::lacZ Fusion Plasmids pPCUR1 and pPCUR2:

[0194] The non-translated pcu promoter region between pcuR and pcuC wasamplified by PCR in order to construct a lacZ fusion to examineregulation of the pcu operon. The reaction contained the following: 0.5μL pPCU1 (0.8 μg/μL), 1 μL primer PCUR1L (10 pmol/μL) (SEQ ID NO:95), 1μL primer PCUR2L (10 pmol/μL) (SEQ ID NO:96), 33.3 mL water, 2.2 μL 25mM Mg(OAc)₂, 1 μL 10 mM dNTPs, 10 μL 5× GC Genomic PCR Reaction Buffer,and 1 μL Advantage-GC Genomic Polymerase Mix (50×). The last fourcomponents were from the Advantage-GC Genomic PCR Kit (Clontech, PaloAlto, Calif.). The reaction was put through the following thermocycles:1 min at 94° C., then 30 cycles of 30 sec at 94° C., 4 min at 68° C.,and incubation at 4° C. overnight. The PCR product was purified usingGeneClean, digested with BamHI and isolated as a 2.4 kb fragmentfollowing electrophoresis on a 0.6% agarose gel. The fragment wasligated to the vector pMC1403 (NCCB no. PC-V3088), which had beendigested with BamHI and dephosphorylated with SAP. The ligation wastransformed into competent Escherichia coli MC1061 cells. Transformantswere selected on LB+Amp (100 mg/L) plates. The orientation of the insertin the vector was determined by SstI and PstI digests, and a plasmidwhere the ribosome binding site and AUG initiation codon from pcuC wasfused to the lac operon was named pPCUR1. A control plasmid with the PCRproduct cloned in the opposite orientation was named pPCUR2.

[0195] Construction of the tmo Plasmid pTMO3:

[0196] The vector pLEX (Invitrogen, Carlsbad, Calif.) was digested withSphI+SstI and ligated to a 6 kb tmo fragment from pTMO1 (FIG. 3)digested with the same enzymes. Ligated DNA was transformed intoEscherichia coli strain G1724 (Invitrogen, Carlsbad, Calif.) andselected on LB+amp (100 mg/L). A plasmid with tmo under thetranscriptional control of the PL promoter was designated pTMO3.

[0197] Construction of the tmo Plasmid pTMO9:

[0198] Plasmid pTMO1 was digested with HindIII+BglII. The 960 bpfragment was isolated and purified with GeneClean, and ligated toHindIII+BglII cut plasmid pSL1180 (Pharmacia, Piscataway, N.J.). Theligation was used to transform competent Escherichia coli XL2 Bluecells, which were then incubated on LB+amp (100 mg/L) plates. HindIIIdigests and NcoI digests of the plasmids from transformants identifiedthose with the correct insert. A correct plasmid was named pTMO6. The960 bp SmaI+HindIII fragment from pTMO6 was isolated and purified asbefore and ligated to the vector pMMB208 (ATTC 37810) which had beendigested with SmaI+HindIII. Competent XL1 Blue cells were transformedwith the ligated DNA and spread onto LB+chl (50 mg/L) plates.HindIII+SstI digests of plasmids from transformants were used todetermine clones with the proper constructs, which were named pTMO7.Next, a 5 kb piece of DNA was isolated from pTMO1 by BglII+BamHIdigestion and inserted into the BamHI site of pTMO7. The ligated DNA wastransformed into competent XL1 Blue cells, which were then spread ontoLB+chl (50 mg/L) plates and incubated at 37° C. until colonies wereapparent. After a few days at 4° C., some of the colonies on the platesdeveloped an indigo-blue color. Plasmids were isolated from indigo-bluecolonies and digested with HindIII to confirm the presence of acorrectly constructed plasmid, which was named pTMO8. The 1.2 kb kanresistance marker from pUC4K (Pharmacia, Piscataway, N.J.) was isolatedby EcoRI digestion, gel electrophoresis, and GeneClean purification. Itwas ligated to EcoRI cut and SAP treated pTMO8, then transformed intocompetent XL1 Blue cells. The correct plasmid from a clone that grew onLB+kan (50 mg/L)+chl (50 mg/L) plates was named pTMO9.

[0199] Construction of the tmo Plasmids pTMO17 and pTMO18:

[0200] A BamHI digest of pTMO11 and a BglII digest of the vector pGV1120(Leemans et al., Gene 19:361-364 (1982)) were electrophoresed on a 0.8%agarose gel. The 6 kb pTMO11 fragment and the vector fragment wereexcised and purified using a GeneClean kit. The two pieces were ligatedtogether, transformed into competent Esvcherichia coli DH5α cells, andspread onto LB+tet (10 mg/L) plates. The plasmids from selected colonieswere digested with HindIII, and one with the correct pattern of bandswas named pTMO17.

[0201] A 7.5 kb BamHI pTMO15 fragment and a BglII fragment from thevector pGV1120 were gel-purified as described earlier. They were ligatedtogether, transformed into Escherichia coli, and plated on LB+tet (10mg/L) plates. HindIII digests of the plasmids from transformants wereused to identify constructs containing the tmo operon, and a correctlyconfigured plasmid was named pTMO18.

[0202] Construction of the Expression Plasmid pMC3 Containing pcu andtmo:

[0203] The pcu operon (pcuC through pcuB) was amplified in a PCRreaction containing 4 μL dNTPs (2.5 mM), primer PCUAMP1 (10 pmol/μL)(SEQ ID NO:93), primer PCUAMP2 (10 pmol/μL) (SEQ ID NO:94), 30.7 μLwater, 0.3 μL pPCU10 (0.3 μg), 2 μL Buffer A, 8 μL Buffer B, and 1 μLElongase (the last 3 components were from the Elongase amplificationsystem (Gibco BRL, Gaithersburg, Md.). The cycles used were as follows:30 sec at 94° C., then 35 cycles of (45 sec at 94° C., 30 sec at 55° C.,5 min at 68° C.), finally 4° C. overnight. The ˜5.5 kb product waspurified using a GeneClean kit, digested with HindIII, isolated from a0.8% agarose gel, and purified again with GeneClean. This fragment wasinserted into a HindIII digested and phosphatased (using CIP) pUC 18vector. The ligation was transformed into competent Escherichia coli XL1Blue cells, and transformants were selected on LB+amp (100 mg/L)+IPTG (1mM)+X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) (50 mg/L) plates.White colonies indicated the presence of an insert in the vector. Thecorrect construct, which was named pPCU14, was found by observingHindIII digest patterns of the plasmids isolated from whitetransformants. Orientation of the inserts was determined by PstI digestpatterns.

[0204] To remove the BamHI site in vector pRK310, the single-strandedends created by a BamHI digest of pRK310 were removed using mung beannuclease. The vector was then allowed to self-ligate and the product waselectroporated into Escherichia coli Electromax DH10B cells. The cellswere spread onto LB+tet (12.5 mg/L) plates to select for-thosecontaining plasmids. BamHI+BglII digests were used to identify clonesthat had the correct construct, which was named pRK310BamKO. This newvector was digested with HindIII and phosphatased with SAP. The 5.5 kbHindIII fragment of pPCU14, isolated as described previously, wasligated into the vector pRK310BamKO. Electromax DH10B cells wereelectropotated with the ligated DNA. Plasmids were isolated from cellsthat grew on LB+tet (12.5 mg/L) plates and were digested, first withHindIII to ascertain the presence of an insert, then with SalI todetermine the orientation of the insert. The correct plasmid was namedpPCU16.

[0205] The 1.2 kb kan resistance marker from the vector pUC4K (PharmaciaBiotech, Piscataway, N.J.) was isolated as an EcoRI fragment in themanner described above, and inserted into the EcoRI site of pTMO1. Theligation was transformed into competent XL1 Blue cells, which were thenspread onto LB+kan (50 mg/L)+amp (100 mg/L)+IPTG (1 mM) plates.Indigo-blue colonies were diagnostic for the presence of the tmo operonbecause tmo-encoded toluene monooxygenase catalyzes, in part, theformation of indigo from indole. The correct construct, which was namedpTMO11, was ascertained through the digestion of the transformingplasmids with BamHI. The 5.9 kb pTMO11 fragment containing the tmo geneswas purified as described previously, and was ligated to BamHI cut andSAP-phosphatased pPCU16. The ligated DNA was electroporated intoElectromax DH10B cells, which were then spread onto LB+tet (12.5mg/L)+IPTG (1 mM) plates. Transformants that carried plasmids with thetmo genes were indigo-blue, as described before. The correct constructwas identified by digestion with PstI, and was named pMC3.

[0206] Construction of the Expression Plasmid pMC4 Containing pcu andtmo:

[0207]Pseudomonas mendocina KR-1 genomic DNA was digested to completionwith EcoRI. The digested DNA was run on a 0.8% agarose gel, and DNAlarger than ˜6 kb was cut out of the gel and purified with GeneClean.Plasmid pUC18 was digested with EcoRI and the ends were phosphatasedwith SAP. The genomic DNA pieces were ligated to the vector, thenelectroporated into Escherichia coli Electromax DH10B cells. The cellswere incubated on LB+amp (100 mg/L)+IPTG (1 mM) plates. Plasmids wereisolated from indigo-producing transformant colonies and digested withEcoRI. The plasmid with the correct digest pattern was named pTMO14. A7.3 kb SmaI fragment from pTMO14 was isolated as before and cloned intothe 2.7 kb HincII cut and SAP treated pUC4K vector. The ligation wasused to electroporate electrocompetent Escherichia coli DH5α cells,which were then incubated on LB+amp (1100 mg/L)+IPTG (1 mM) plates.BamHI digests were performed on plasmids from indigo-blue colonies fromthe plates. The correct construct, which had the tmo operon flanked byBamHI sites, was named pTMO15.

[0208] The 7.3 kb BamHI pTMO15 fragment was isolated as before andinserted into the BamHI site of pPCU18. This ligated DNA waselectroporated into electrocompetent Pseudomonas putida DOT-T1 C5aAR1cells, which were incubated on LB+strep (50 mg/L)+indole (1 mM) plates.Some of the plates also had a drop of toluene added to the inside of thetop lid. They were all incubated at 30° C. overnight. PstI digests ofplasmids from transformants identified one that had both pcu and tmooperons, and this clone was named pMC4 (FIG. 4).

Example 4 Production of p-Cresol from Toluene in Escherichia coli

[0209]Escherichia coli strain JM105 harboring plasmid pTMO1, whichplaces tmo expression under control of the lac promoter, was grown underinducing conditions in the presence of 1 mM IPTG, or under non-inducingconditions in the absence of IPTG. Escherichia coli strain G1724harboring plasmid pTMO3, which places tmo expression under control ofthe PL promoter, was grown under inducing conditions in the presence of100 mg/L tryptophan, or under non-inducing conditions in the absence oftryptophan.

[0210] Induced or non-induced cell samples were resuspended in minimalmedium at a concentration of 100 mg/mL. To 26 mL of minimal medium in a125 mL sealed flask was added 4 mL of the cell suspension, and 1 mL oftoluene placed in a center well. Following a 36 h incubation 15 mL ofthe cells were acidified, extracted with ethyl acetate, and analysed byGC/MS. Table 2 shows that p-cresol is produced when induced cellsharboring either plasmid pTMO1 or plasmid pTMO3 are incubated in thepresence of toluene. In contrast, in the absence of induction of tmousing either plasmid, no p-cresol is detectable. TABLE 2 Plasmid InducerGC Peak Area pTMO1 IPTG 1.45 × 10⁶ pTMO1 None 0 pTMO3 Tryptophan 5.05 ×10⁵ pTMO3 None 0

Example 5 Bioconversion of Toluene to p-cresol in Pseudomonas putidaATCC 29607

[0211]Pseudomonas putida ATCC 29607 was transformed with pTMO9 andpPCU12, grown at 30° C. and 250 rpm in medium A (Table 3). At an OD₆₀₀of 1.98 (16 h) cells were harvested and washed in MM#4 medium (Table 4).(Trace elements found in both medium A and MM#4 Medium are listed inTable 5.) PHBA production was carried out in 125 mL sealed flasks in 5mL MM#4 medium that contained 0.5 OD₆₀₀ cells, 0.05 mM MgSO₄, 2 mMglucose, 1 mM IPTG, 0.1 M HEPES buffer pH 7.5-8.0 and 60 ppm toluene.The flasks were incubated shaking at 250 rpm and 30° C. A non-inducedcontrol did not have IPTG added. Samples were incubated for 6 h, and thepresence of p-cresol detected by HPLC. In the presence of IPTG 0.93 mMp-cresol was present after 6 h, compared to 0.135 mM in the non-inducedsample. TABLE 3 Medium A per L Special Conditions KH₂PO₄ 1.2 g (NH4)₂SO₄3 g glucose 7 g/L sterilized separately MgSO₄.7H₂0 0.15 g trace elements10 mL sterilized separately HEPES 0.05 M yeast extract 1 g sterilizedseparately

[0212] TABLE 4 MM #4 Medium trace elements 10 mL yeast extract 0.48 gMgSO₄.7H₂0 10 mM NaKPO₄ 25 mM DD H₂O 1 L PH 7.2

[0213] TABLE 5 Trace Elements in Medium A and MM#4 g/L citric acid 10CaCl₂.2H₂O 1.5 FeSO₄.7H₂O 2.8 ZnSO₄.7H₂O 0.39 trace elements Medium Aand MM#4 CuSO₄.5H₂O 0.38 CoCl₂.6H₂O 0.2 MnCl₂.4H₂O 0.3

Example 6 Identification and Sequence of a tmo Regulatory Region

[0214] Detection of a Regulatory Sequence:

[0215] Plasmids pTMO17 and pTMO18 differ in the amount of tmo sequenceinformation that is present. Plasmid pTMO17 contains the six toluenemonooxygenase genes tmoA-F. Plasmid pTMO18 also contains tmoA-F, but inaddition has 1326 bp of DNA sequence information upstream from thetranslational initiation codon of tmoA. Plasmids pTMO17 and pTMO18 weretransformed separately into Pseudomonas putida DOT-T1 C5aAR1 andselected on LB+strep (100 mg/L). Colonies were inoculated into 25 mLLB+1 mM indole+strep (100 mg/L) and shaken in a 125 mL baffel flask at200 rpm and 30° C. until indigo production occured. A 5 mL sample ofcell suspension was extracted twice with an equal volume of ethylacetate to solubilize the indigo, the two extracts were combined and theabsorption at 600 nm recorded. A standard curve prepared with pureindigo in ethyl acetate was used to determine amounts in cell extracts.

[0216] TMO enzyme assays were carried out in a separate experiment usingthe same plasmids and strain. TMO was measured spectrophotometricallyusing a coupled assay, linking phenazine ethosulfate (PES) oxidation toreduction of 2,6-dichlorophenol-indophenol (DCPIP) as measured by adecrease in absorption at 600 mm (E_(600 nm)=21,000 M⁻¹ cm⁻¹). The assaywas initiated by the addition of enzyme to a 2.0 mL reaction mixturecontaining 0.67 μmol PES, 0.1 μmol DCPIP, 1.0 μmol toluene andsaturating levels of purified p-cresol methylhydroxylase.

[0217] Table 6 shows that the presence of additional DNA upstream oftmoA enhances the level of TMO activity, which leads to a considerableimprovement in indigo prduction. TABLE 6 Plasmid TMO activity Indigoproduced (mg/L) pTMO17 0.7 2.5 pTMO18 1.3 88.0

[0218] Sequence of tmoX and its Upstream Promoter Region:

[0219] The DNA upstream of tmoA was sequenced with synthetic primers(SEQ ID NO:80 to SEQ ID NO:90) according to standard methods. Thecomplete sequence of the DNA has the sequence found in SEQ ID NO:91.Encoded within the sequence is a protein, TmoX, with the initiatormethionine at nucleotide 192 and a TAA translation terminator atposition 1560. The predicted amino acid sequence of TmoX is given as SEQID NO:92 and its nucleotide sequence is in SEQ ID NO:103. TmoX has an81% identity (87% similarity) in its predicted amino acid sequencecompared to that of the TodX protein of Pseudomonas putida DOT-T1 (Table1). TodX has been described as an outer membrane protein that may beinvolved in facilitating the delivery of exogenous toluene inside cells(Wang et al., Mol Gen. Genet. 246:570-579 (1995)), but has also beenlinked to the signal transduction process which results in specificresponse of a tod promoter to toluene (Lau et al., Proc. Natl. Acad.Sci. USA 94:1453-1458 (1997)).

[0220] The tmoX promoter was identified by primer extension using a23-mer oligonucleotide (SEQ ID NO:97) complementary to the DNA codingstrand. The first nucleotide of the primer corresponded to a nucleotide200 bp downstream from the A of the ATG initiation codon of the tmoXgene. Pseudomonas mendocina KR-1 was grown overnight in M9 minimalmedium with 10 mM citrate as the sole carbon source. To 200 mL of freshmedium was added 5 mL of overnight culture to give an initial OD ofabout 0.2 at 660 nm. The culture was incubated at 30° C. on a rotaryshaker to an OD of 0.8 at 660 nm. Aliquots of 20 mL were supplementedwith either 1 mM p-cresol, toluene in the gas phase, or a control withno additions. Samples were used for RNA isolation at 30, 60 and 180 minafter addition of the effector.

[0221] The primer (SEQ ID NO:97) was labeled at its 5′ end using³²P-γ-ATP and polynucleotide kinase. To 30 μg of total RNA for eachsample were added 10⁵ CPM of labeled primer, which was extended usingreverse transcriptase. The resulting cDNA was separated on aurea-polyacrylamide sequencing gel. In addition, the labeled primer wasused to establish a sequencing ladder to facilitate the identificationof the transcription initiation point. It was established that the 260base cDNA product positioned the tmoX transcription initiation point asa G located 60 bp upstream of the A of the ATG translation initiationcodon of tmoX. Analysis of the region upstream of the ATG codon showsthe presence of a prokaryotic Shine-Dalgarno sequence. Also noted is thepresence of −10 and −35 sequences upstream of the transcript initiationsite, each positioned respectively at bp 124-128 and bp 101-105 in SEQID NO:91. A putative TodT motif is to be found at bp 30-46 in SEQ IDNO:91.

[0222] By comparing the amounts of cDNA obtained under differentinduction regimes, it was found that growth in the presence of tolueneled to a 20-fold increase in tmoX mRNA compared to growth on citrate,with a maximal level observed 30 min after exposure to the solvent. Thetranscription of tmoX was also induced by the presence of p-cresol, withmaximal levels also at 30 min, followed by a decrease in signalintensity probably related to exhaustion of the inducer in the culturemedium.

Example 7 Regulation of pcu Expression by PCUR

[0223] The pcuC::lacZ fusion plasmid pPCUR1, and the control plasmidpPCUR2, were transformed into Escherichia coli MC1061. Plasmids pPCUR1and pPCUR2 also encode PcuR and amp resistance. Cultures were grownovernight in flasks shaking at 37° C. in M9 minimal medium containing 1%glucose and 50 mg/L amp. In addition, some flasks also containedintermediate compounds of the toluene to PHBA pathway, including tolueneand PHBA. The following were added at a concentration of 1 mM toseparate flasks prior to overnight incubation: p-cresol,p-hydroxybenzylalcohol, p-hydroxybenzaldehyde and PHBA. Toluene (5 μL)was added to the gas phase of a 125 mL sealed flask. The overnightcultures were treated with chloroform and SDS, and assayed forβ-galactosidase as described in J. H. Miller in A Short Course inBacterial Genetics (Cold Spring Harbor Laboratory Press: Cold SpringHarbor, N.Y.; 1992).

[0224] Table 7 shows that when using plasmid pPCUR1 there is noinduction of the pcuC:: lacZ gene fusion when toluene or PHBA arepresent, neither of which are substrates for enzymes encoded by the pcuoperon. In contrast, the presence of p-cresol, p-hydroxybenzylalcohol orp-hydroxybenzaldehyde all lead to significant induction of pcuC::lacZ,and all three compounds are substrates for the two enzymes encoded bythe pcu operon i.e. PCMH and PHBAD. In the control plasmid pPCUR2, inwhich the pcuC gene is incorrectly orientated for expression, thepresence of p-cresol does not lead to expression of β-galactosidaseactivity. TABLE 7 Plasmid Inducer β-galactosidase units pPCUR1 toluene0.55 pPCUR1 p-cresol 19.53 pPCUR2 p-cresol 0.05 pPCUR1p-hydroxybenzylalcohol 5.65 pPCUR1 p-hydroxybenzaldehyde 9.70 pPCUR1PHBA 0.06

Example 8 Activity of Plasmid-Encoded Enzymes in Pseudomonas putidaDOT-T1

[0225] Cells were grown in Medium A with the appropriate antibiotic inshake flasks at 30° C. (200 rpm). When the glucose had been depleted,the induction phase was initiated by addition of toluene and/orp-cresol. Three consecutive additions of inducer were made, eachseparated by one hour. For induction with IPTG, the compound was addedat a concentration of 1 mM. Cells were collected by centrifugation,washed once with phosphate buffered saline and stored at −80° C. untilassay.

[0226] TMO was measured spectrophotometrically using a coupled assay,linking phenazine ethosulfate (PES) oxidation to reduction of2,6-dichlorophenol-indophenol (DCPIP) as measured by a decrease inabsorption at 600 nm (E_(600 nm)=21,000 M⁻¹ cm⁻¹). The assay wasinitiated by the addition of enzyme to a 2.0 mL reaction mixturecontaining 0.67 μmol PES, 0.1 μmol DCPIP, 1.0 μmol toluene andsaturating levels of purified p-cresol methylhydroxylase (PCMH).

[0227] p-Cresol methylhydroxylase (PCMH) activity was measuredspectrophotometrically using a coupled assay, linking phenazineethosulfate (PES) oxidation to reduction of2,6-dichlorophenol-indophenol (DCPIP) as measured by a decrease inabsorption at 600 nm (E_(600 nm)=21,000 M⁻¹ cm⁻¹). The assay wasinitiated by the addition of enzyme to a 2.0 mL reaction mixturecontaining 0.67 μmol PES, 0.1 μmol DCPIP and 1.0 μmol p-cresol. Thisassay was also used to measure toluene monooxygenase (TMO) activity bysubstituting 0.5-1.0 μmol toluene into the reaction mixture and by theaddition of saturating levels of purified p-cresol methylhydroxylase(PCMH).

[0228] p-Hydroxybenzoate dehydrogenase (PHBAD) activity was measuredspectrophoto-metrically using a reaction mix containing 600 nmol NADP⁺,40 nmol p-hydroxybenzaldehyde, 1.0 mL of 50 mM glycine-NaOH (pH 9.6),and an appropriate amount of enzyme. Enzyme activity was determined byan increase in absorbance at 330 nm. A unit of activity is the amount ofenzyme required to oxidize 1.0 μmol of p-hydroxybenzaldehyde per min(E_(330 nm)=28,800 M⁻¹ cm⁻¹).

[0229] p-Hydroxybenzoate hydroxylase (PHBH) was assayedspectrophoto-metrically by following the oxidation of NADPH. Thereaction mixture contained 250 nmol NADPH, 700 nmol p-hydroxybenzoate,an appropriate amounts of enzyme, and 50 mM Tris-HCl buffer (pH 8.0) togive a final volume of 1.0 mL. A unit of activity is the amount ofenzyme required to oxidize 1.0 μmol of NADPH per min (E_(340 nm)=6,200M⁻¹ cm⁻¹).

[0230] Enzyme assays for PCMH and PHBAD demonstrate that both of thePseudomonas mendocina pcu enzymes are expressed in Pseudomonas putidastrain DOT-T1 (Table 8). In addition, it is noteworthy that expressionof pcu is superior when using its native promoter in plasmid pMC4compared to the use of a lac promoter in plasmid pMC3. This is also truefor TMO, where greater activity is seen when using the endogenous tmopromoter in pMC4 when compared to the lac promoter in plasmid pMC3.TABLE 8 Plasmid Promoter Inducer TMO PCMH PHBAD pMC3 lac IPTG 0.76 0.74pMC4 pcu or tmo Toluene 18.2 13.5 9.0 pPCU18 pcu p-cresol 0 16.1 3.6none — — 0 0.29 0.05

Example 9 Production of PHBA from p-Cresol by Pseudomonas putida ATCC29607 Transformed with a pcu Expression Plasmid

[0231] The mobilizing Escherichia coli strain SI 7-1 was used tointroduce the pcu expression plasmid pPCU12 into Pseudomonas putida ATCC29607 by conjugation. A single colony of S17-1 having the plasmid pPCU12was inoculated in 20 mL LB medium and grown at 37° C. to log phase.Another colony of Pseudomonas putida ATCC 29607 was inoculated in 20 mLLB medium and incubated at 30° C. and grown to log phase. The cells ofboth cultures were washed twice with LB medium and resuspended in LBmedium. S17-1 cells harboring pPCU12 and Pseudomonas putida ATCC 29607were mixed at a ratio of 1:4 and were plated on agar plates of LBmedium. The plates were incubated at 30° C. for 8 h. The cells werecollected and then plated on agar plates containing phosphate buffer, 1mM succinate, 10 mM strep and 25 mg/L kan, and kan resistant colonieswere selected. Transformants, or a non-transformed control strain, weregrown in 15 mL M9 minimal medium containing 1% glucose, 5 mM p-cresol,10 mM MgSO₄, tet (15 mg/L) in 125 mL flasks at 30° C. and 225 rpm.Samples were removed at the indicated timepoints and analyzed by HPLCfor the presence of p-cresol and PHBA. In a plasmid-free controlPseudomonas putida strain failed to convert 3.3 mM p-cresol to PHBA(<0.007 mM). In contrast, Pseudomonas putida harboring plasmid pPCU12produced 0.793 mM PHBA during an overnight incubation (Table 9). PHBAproduction is, therefore, a new attribute of Pseudomonas putida ATCC29607 when transformed with and expressing pcu. TABLE 9 Strain Time (h)PHBA (mM) p-cresol (mM) control 2 <0.007 3.3 control 5 <0.007 3.4control 16 <0.007 3.2 pPCU12 2 0.141 2.8 pPCU12 5 0.284 2.8 pPCU12 160.767 2.2

Example 10 Increased Rate of Production of PHBA from p-Cresol byPseudomonas mendocina Harboring a pcu Expression Plasmid

[0232] Plasmid pPCU12 was transferred by conjugation from Escherichiacoli S17-1 to Pseudomonas mendocina KRC16KDpobA51 as described earlier.Transformants, or a non-transformed control strain, were grown in 15 mLM9 minimal medium containing 1% glucose, 5 mM p-cresol, 10 mM MgSO₄, tet(15 mg/L) in 125 mL flasks at 30° C. and 225 rpm. Samples were removedat intervals of 2, 5 and 16 h and analyzed by HPLC for the presence ofp-cresol and PHBA. The Pseudomonas mendocina KRC16KDpobA51 strain has afunctional chromosomal pcu operon, but also has inactivated pobA genesto enable PHBA to accumulate. In the presence of the pPCU12 expressionplasmid in Pseudomonas mendocina KRC16KDpobA51, PHBA accumulates morerapidly to give a concentration of 1.57 mM during the first 5 hincubation, compared to 0.526 mM for the control Pseudomonas mendocinastrain alone (Table 10). TABLE 10 Strain Time (h) PHBA (mM) p-cresol(mM) control 2 0.185 3.2 control 5 0.526 2.8 control 16 4.02 0.48 pPCU122 0.7 2.8 pPCU12 5 1.57 2.2 pPCU12 16 4.84 0.08

Example 11 Production of PHBA from p-Cresol by Agrobacterium rhizogenesATCC 15834 Transformed with a pcu Expression Plasmid

[0233]Agrobacterium rhizogenes ATCC 15834 was grown in nutrient broth at30° C. and cells harvested during logarithmic growth. The cells weremade electrocompetent by washing three times in water, centrifuging at6000 rpm after each wash. Either the plasmid vector pGV1120 (Leemans etal., Gene 19:361-364 (1982)) or pMC4 were electroporated into the cellsusing 1 mm gap cuvettes at 1.44 kv. Cells were spread on LB platescontaining 50 mg/L strep and incubated at 30° C. Transformants harboringthe pGV1120 vector, or the pcu expression plasmid pMC4, were grown for24 h in nutrient broth containing 50 mg/L strep, 10 mM MgSO₄, and 1 mMfully-deuterated p-cresol. PHBA was extracted from boiled cells withether and concentrated by evaporation. Gas chromatography/massspectrometry was used to show that the PHBA formed 0.10 (1.4 μM)contained 4 deuterium atoms. This experiment proves that it was derivedfrom the p-cresol present during culture of the cells.

Example 12 Production of PHBA from Toluene by Pseudomonas mendocinaTransformed with Plasmid pMC3 (pcu⁺ tmo⁺)

[0234]Pseudomonas mendocina KRC16KDpobA51 was transformed with plasmidpMC3 and selected on LB+tet (12.5 mg/L) plates at 30° C. The procedurefor cell growth and toluene production was similar to that described inExample 9. The test cultures have 1 mM IPTG present at the growth andPHBA production stages in order to induce transcription from the lacpromoter. No IPTG was added to the control cultures. Samples were testedfor PHBA by HPLC at 1, 2, 4 and 6 h intervals. Table 11 shows that PHBAis produced by induced and non-induced cultures, but with IPTG-treatedcells production started earlier, and approached levels that were withinthe maximum expected based on the amount of toluene added in the flasks.TABLE 11 PHBA (mM) Time (h) +IPTG −IPTG 1 0.147 0.131 2 0.438 0.180 44.985 1.230 6 7.442 4.172

Example 13 Generation of Stable ΔtodC Deficient Pseudomonas putidaDOT-T1E Strains

[0235]Pseudomonas putida DOT-T1E (CECT 5312) grows on toluene via thetoluene dioxygenase pathway (Mosqueda et al., Gene 232:69-76 (1999)).The use of this strain for PHBA production from toluene requires itsinactivation. In order to generate a mutant deficient in toluenemetabolism in DOT-T1E strain, a deletion of the todC1 gene in the todoperon was carried out. FIG. 5 illustrates the strategy used and therelevant constructions. The entire DOT-T1 tod operon (Mosqueda et al.,Gene 232:69-76 (1999)) is contained in two plasmids: todXF genes borneby pT1-4, and todC1C2BADEGIHST genes borne by pT1-125. The approximately4.5 kb EcoRI/XcaI fragment of pT1-125 which extends from todC1 to todDwas cloned at the EcoRI/SmaI sites of a pUC18 Not derivative (de Lorenzoand Timmis, Methods Enzymol. 235:386-405 (1994)) that lacked the BamHIand HindIII at the multicopy cloning site to give plasmid pMIR17. The1.8 kb SspI/EcoRI fragment of pT1-4 containing todXF was cloned at theEcoRI site of pMIR17 and the plasmid pMIR20 was obtained. (The uniqueNotI site present in the SspI/EcoRI fragment was removed beforecloning). Most of the 3′-half end of todF, the entire todC1 gene, andthe 5′-end of todC2 were removed from pMIR20 as a 1.6 BamHI/HindIIIfragment. A 2.2 kb fragment containing the Ω/km cassette (Fellay et al.,Gene 52:147-154 (1987)), encoding resistance to kanamycin, was cloned atthe same position which rendered the pMIR22 plasmid. pMIR30 was obtainedas the result of the subcloning in pKNG101 of the NotI fragments, whichcontained the region corresponding to the ΔtodC1 and the Km resistanceof pMIR22. pKNG101 is a suicide vector in Pseudomonas which confersconditional lethality in the presence of sucrose (Kaniga et al., Gene109:137-141 (1991)). pMIR30 was used to replace the todC1 gene in thechromosome of Pseudomonas putida DOT-T1E with a deleted version byhomologous recombination and a toluene minus DOT-T1E derivative wasobtained called ΔtodCkm. The absence of todC1 gene in the chromosome ofthe toluene minus isolate was confirmed by PCR with specific primers andin Southern blot.

[0236] The stability of the mutant unable to use toluene as the solecarbon-source was tested. The results can be summarized as follows: i)after 90 generations of growth on LB medium under non-selectiveconditions, i.e. in the absence of antibiotic markers, 100% of cellswere resistant to kanamycin and unable to grow in toluene; ii) no growthwas observed in M9 liquid minimal medium with toluene as the solecarbon-source; i.e. revertants were undetectable after one week inflasks with 10 mL cultures which had been inoculated with 10⁷ CFU/mL);iii) the reversion rate determined as the re-acquisition of the abilityto grow on toluene was undetectable (lower than 10⁻⁹ by the platingtechnique).

Example 14 Cloning of the Pseudomonas putida pobA Gene

[0237] The pobA gene encodes the enzyme para-hydoxybenzoate hydroxylaseand converts PHBA into protocarechuate. Production of PHBA requires thatits metabolism through the pobA pathway be impaired. To this end, pobAwas first cloned, then it was inactivated it in vitro and the mutationtransfered to the chromosome of ΔtodKm. To clone Pseudomonas putida pobAgene, a Pseudomonas putida KT2440 (ATCC 47054) built in the tetracycline(Tc)-resistant pLAFR3 cosmid (Rodriguez-Herva et al., J. Bacteriol.178:1699-1706 (1996)) was used for the complementation of thePseudomonas mendocina KRC16KDpobA51 (ATCC 55885). In this strain bothpobA genes are inactivated and so it is unable to grow inp-hydroxybenzoate (WO 98/56920). Upon triparental mating with E. coliHB101 (pLAFR3::genebank), E. coli (pRK600)—a helper strain- andPseudomonas mendocina #303, Tc^(R) Pseudomonas mendocina exconjugantsable to grow on p-hydroxybenzoate as the sole carbon source wereselected. The chimeric cosmids of these clones were isolated, theirrestriction pattern established and analyzed in Southern blot againstthe Pseudomonas mendocina pobA1 gene. A 6 kb BamHIII/EcoRI hybridizationband common to all cosmids was found and cloned in pUC19 to yieldpMIR18. Plasmid pMIR18 was used as the target for “artificial” in vitrorandom transposition, which was carried out with the Primer IslandTransposition Kit (PE Applied Biosystems). A battery of plasmidscarrying the AT-2 transposon at different positions was generated and E.coli DH5α cells were electroporated with the heterogeneous mix ofplasmids. pobA and pobR genes were identified by sequencing fromspecific present at the extremes of the transposable element in pMIR27(FIG. 6). The following primers were used: 1383-1399 oligo pobA1 (+) 5GCTTCCACGGTATCTCG 3; (SEQ ID NO:104) 1359-1343 oligo pobA1 (−) 5CAGTCAATCCGCTGCAC 3; (SEQ ID NO:105) 1732-1751 oligo pobA2 (+) 5GCAGTATGGTCACCTGTTCC 3; (SEQ ID NO:106) 1728-1710 oligo pobA2 (−) 5GGTTCGACCACCAGGCTAC 3; (SEQ ID NO:107) 1162-1180 oligo pobA3 (+) 5GGATCTCAAAGCCCTGACC 3; (SEQ ID NO:108) 963-983 oligo pobA4 (+) 5TGCTGCACAAGGCCGGTATCG 3; (SEQ ID NO:109) 1945-1925 oligo pobA4 (−) 5GGTCATGAACCAGCTGAAGCG 3; (SEQ ID NO:110) 742-760 oligo pobR2 (−) 5CCTGTCCGTTAATCGAACG 3. (SEQ ID NO:111)

Example 15 Generation of p-Hydroxybenzoate Minus Derivative ofPseudomonas putida T1-E ΔtodCkm

[0238] To knock-out the pobA gene in the chromosome of the toluene minusPseudomonas putida ΔtodCkm strain, plasmid pMIR31 was generated with apobA inactivated copy (FIG. 6). Plasmid pMIR31 bore Pseudomonas putidaKT2440 pobA gene interrupted by the interposon Ω/Sm. This chimericplasmid is a suicide vector in Pseudomonas and was used as a deliverysystem for gene replacement of the wild type pobA allele for aninactivated copy by homologous recombination.

[0239]Pseudomonas putida ΔtodCkm cells were electroporated with pMIR31and after high voltage pulse, cells were incubated in SOC medium for twoh at 30° C., then centrifuged and the pellet incubated overnight on anLB-agar plate. Finally, Sm-resistant transconjugants were selected on LBplates with Km, 25 μg/mL, and Sm, 150μ/mL. This selection mediumpermitted the growth of the clones resulting from a single cointegrationevent of pMIR31 in the host chromosome, as well as an eventuallysuccessful gene replacement after the resolution of the cointegrate. Twohundred Km^(r) Sm^(r) colonies were tested for their ability to grow onp-hydroxybenzoate as the sole carbon source and for piperacillinresistance (Pip^(r))—the marker of the pMIR31 plasmid that allowed oneto confirm the cointegration of the host chromosome of the plasmid. Twoof the clones were Km^(r) Sm^(r) p-hydroxybenzoate—Pip^(s) glucose⁺ wasused to confirm the successful allelic exchange of the wild-type pobAgene for the inactivated copy confirmed by Southern blot. The doublemutant was called Pseudomonas putida todCKmpobA::Sm.

Example 16 Recruitment of Pseudomonas mendocina KR1 TolueneMonooxygenase/p-Cresol Utilization Pathways in Pseudomonas putidaΔtodCKmpobA::Sm (Construction of a miniTn5Tctmo/pcu Transposon andProduction of PHBA)

[0240] A transposon was constructed based on a miniTn5Tc withPseudomonas mendocina tmo/pcu genes which permitted integrating thesecatabolic genes in the chromosome of the double mutant Pseudomonasputida ΔtodCKmpobA::Sm and so produced p-hydroxybenzoate from toluene.The scheme of the construction of the transposon is shown in FIG. 7. The7.5 kb BamHI fragment of pMC4 containing the tmoXABCDEF genes wassubloned at the same site in the polylinker of pUC19, generating theplamid pMIR32. The 7.6 MluI/NheI fragment of pPCU17 containing thepcuRCAB genes was subcloned at the HindII/XbaI sites of pUC18NotI. Inthe plasmid so generated, pMIR40, the 7.4 kb BamHI fragment of pMIR32containing the tmo operon was cloned at the BamHI site.

[0241] Then the 15 kb NotI fragment containing pcu and tmo genes wascloned at the unique NotI site of pUT/Tc (de Lorenzo and Timmis, MethodsEnzymol. 235:386-405 (1994)) generating the plasmid pMIR44 (the uniqueNotI site of pUT/Tc is located within the transposable element miniTn5Tcborn by the plasmid pUT which is suicide in Pseudomonas). The transposonwas delivered in the chromosome of the double mutant Pseudomonas putidaΔtodCKmpobA::Sm via a triparental mating with CC118λpir (pMIR44) as adonor and HB101 (pRK600) as a helper strain. Exconjugants Km^(r) Sm^(r)Tc^(r) were selected with a rate of 5×10⁻⁸. The presence of theminiTn5Tctmo/pcu transposon was confirmed in the Tc^(r) exconjugants byPCR-amplification of the tmoA gene. This strain produces more than 2 g/LPHBA when grown with glucose in the presence of toluene.

Example 17 Construction of a pobA Mutant of Pseudomonas putida KT2440and Recruitment of miniTn5Tctmo/pcu

[0242] Plasmid pMIR31 was also used to replace the wild-type pobA geneof Pseudomonas putida KT2440 with a mutant allele as it was carried outwith Pseudomonas putida ΔtodCKm. The resolution of the merodiploidcolonies was tested for Sm resistance and Pip sensitivity. One out of100 colonies exhibited this character and was unable to grow onp-hydroxybenzoate as the sole carbon source. The allelic exchange wasfurther confirmed on Southern blot.

[0243] The catabolic genes tmo/pcu were recruited in Pseudomonas putidapobA through a triparental mating with CC118λpir (pMIR44), HB101(pRK600) and Pseudomonas putida pobA, as it was previously conducted forthe recruitment of the miniTn5Tctmo/pcu in Pseudomonas putidaΔtodCKmpobA::Sm. Nevertheless, this strain only produced trace amountsof PHBA. However production of PHBA was achieved when the regulatorytodST genes of Pseudomonas putida DOT-T1E (SEQ ID NO:112; GenBankAccession Number Y18245; Mosqueda et al., Gene 232:69-76 (1999)) wereintroduced in the strain after subcloning in plasmid pBBR1-MCS5. Thisstrain produced PHBA up to 10-15 mM in 250 mL flasks with 3 mL culturecontaining about 10⁸ cells/mL and incubated at 30° C. on an orbitalplatform operated at 200 strokes per min. This example indicates thatthe regulatory genes of the tod pathway induce the tmo pathway.

[0244] The heterologous TodST proteins that control the induction oftoluene dioxygenase pathway, are able to induce high levels ofexpression from the tmo pathway genes, and are useful tools to mediateexpression of the catabolic tmo genes and PHBA production in anyorganism that does not possess these genes. Previously, Lau andco-workers (Proc. Natl. Acad. Sci. USA 94:1453-1458 (1997)) have shownthat the two regulatory genes from Pseudomonas putida F1, todS and todT,are members of a two-component signal tranduction family of bacteriauses a histidine-asparate phosphorelay circuit to sense environmentalchanges. The genes in the instant invention are 95-100% homologous tothe tod genes in Pseudomonas putida F1.

1 112 1 6491 DNA Pseudomonas mendocina KR-1 1 tcactccccc ttgagccggtagctgatctg cgcgcgactc atgcccaaca tctgcgccgc 60 cgcggtgagg ttgccgccggtgcgttccag ggcaaggtgc accaggcgct gctcgatctc 120 cttcagtgat gtgcctagtacccggtcgcg cccggcgagg aaggcctgca ggttggccag 180 ccccagctca accggttcatgctcctcgac aaccacctca gcccgcgctt gcggttcgcc 240 gccgacggca tccagacggccttcggcggt caggccgatg ccgctggagc gaagtggctc 300 gccggctttt gccaggtgcaccaggtcgat cagctcgcca ctgcctgcgg cgatcacgcc 360 gcgctcgatc aggttctgcagctcacggat attgccgggg aagcggtagg tcagcagcgc 420 gttgaccagc cgcgtgctgaaacccagggg tttgagccca tggcgcgcac tgaacttgcg 480 caggaagtag ctcatcaggagcgggatgtc ctcacggcgc tcgcgcaggg gcggcagatg 540 gatggggaac acgttcagccggtacagcag gtcctcgcgg aagcgcccgg cctcgacctc 600 tcggcgcagg tccagattggtggcggcgat caccctcaca tccaccggga tcgccgaggt 660 accacctacc cgctcgatctcgccctcctg cagcacccgc aggatcttgc tctgggcgct 720 gaggctcagg gtggcgatctcgtcgaggaa cagggtgccg cccttggccc gctcgaagcg 780 ccccgggcgg gaacggtcggcgccggtgaa ggcaccgcgc tccacgccga acagttcggc 840 ttccagcaga gtttccggcaacgccgcgca gttgagcgcc accaacggcg tttggcggcg 900 cgggctggcc tggtgcagggtgcgcgcgaa gagctccttg cccacccccg attcaccggt 960 cagcagtacg gtggcctgggtcgacgcaac gcggtagagc tgctggctgg cggcgacaaa 1020 ggcggcggaa atgcccaccatggcctggtc ctcgggcggc tcatccagat cggccatttc 1080 cgtctcgtct gccgagccgtaggtgctccg gctgaggaag tcgctggcat ccaggtgggc 1140 caggtcggtg tcgatgtcctcccactgctc cgccggcttg ccgacgatgc ggcacgccga 1200 atggcccatg cagcggcattcctgctcgcg gaacaccacc aggcgcccca gcagggagga 1260 ggtgtagccg ctggcgtagcccacttccat ccagcaggcc ggttcgctgc ccagcccgta 1320 gctggcgatg tgctcgtcggcttccaggga gttgtgccag aagaattcgg aatagaaatg 1380 cccgatgctg gagtcgatgtcgaagcgcac cacttccacg ttcaccatgc cctccagcat 1440 gtgcaggcgc gggcctgcgctgtagaggct ggcgtggtcg ccctcgggcc actgcgcgct 1500 gacctgagcg gcatccctcgttccggcctg ccagccaatg cgggtcagaa ggccacgggc 1560 cttgtcgagg ccgagggcttccaccaactc gcgacggatg gcgccgaagg cggccccctg 1620 cagcagcatc atgcgctggccgcagagcca gatattgcca tcctggggcg cgaaggcgac 1680 ggtctccgcc agttgctcggccgagggcag cccgctgctg ccgaactggt tggcctggtc 1740 gacgatcagg ctcttctgccggccgagcaa ctgcttgagg aattcatccc ccatgctgcg 1800 gccgggattg ctcgagggtttgcgagtcat ggtcatgggg cgggaggtag gaacaatgtt 1860 attcagtatg cccgtgtgaaatggccggtc aattggccct tgccatcacc caataatcgc 1920 ccaacctctt gcagaccactccggagaagt ttctgcgccc cggagacttc tctgaagaaa 1980 aatcggcgcc aaccctcccgcaagcccccc atgcgtccgc tccgcattcc ccaaaaaaac 2040 gtaaccaatt gttttacaaataaaaaatag aagaaagaag gattggcacg gtagttgtta 2100 aaggacaggg gcgtgcacccaagacaataa caacacaggt aacgacccta tgaaccgctt 2160 cccatcgcca atccattccgcttgcccacc cgcaccacgg cttcgttgtt gaccctcaac 2220 cgtacctcca caggaacggcgcccgcgcgt cttgcctgac gtatcgccac gcgcccgtgt 2280 aaccaccggc tcgccgccactggcagcctt ccgcgcaaac aagagagaac ccatggacac 2340 cacccgccct gcctaccagaacctcgagct ccaacctctc gccgggcaat ggcgcgccgg 2400 cagtagcggt cgcccgttggaggtcttcga cccctacaac gacgagctgc tattgcgcat 2460 cgccctggcc agccgcgaagacctcgacgc agcctaccgc aaggcccgcg acagccagcg 2520 ggagtgggcg accacggcgccggccgagcg cgcccgggtg ctgctggaag cggtgaagat 2580 cttcgatgag cgccgcgaggagattatcga ctggatcatc cgcgagtccg gcagcacccg 2640 catcaaggcg cagatcgaatggggcgccgc ccgcgccatc accctggagt cggccagcct 2700 gccgaatcgc gtgcacgggcgcatcatcgc ctccaacatc tccggcaagg agagccgcgt 2760 gtaccgcgcg cccctgggcgtgatcggcgt gatcagtccg tggaacttcc ccctgcacct 2820 cactgcccgc tccctggccccggccctggc cctgggcaat gccgtggtgg tcaagccggc 2880 cagcgacacc ccgatcaccggtggcctact gctggcgcgc atcttcgaag aagccggcct 2940 gccggcgggc gtgctcagcgtggtggtggg ttcgggcgcg gagattggtg acgccttcgt 3000 cgagcacccg gtgcccgccctcatttcctt caccggctcc actcaggtgg gccgcaacat 3060 cggccgcatc gccagcggcggtgagcacct caagcacgtg gcgctggaac tgggcggcaa 3120 cagcccgttt gtggtcttggccgatgccga cgtggagcag gcggtgaatg cggccgtggt 3180 cggcaagttc ctgcaccagggccagatctg catggcgatc aaccgcatta tcgtcgagca 3240 gcctttgctg gaagatttcacccgccgctt cgtcgagcgc gtcaaggccc tgccctatgg 3300 cgacccgagc aagccggggaccgtggtcgg tccggtgatc aacgccaggc agctggccgg 3360 tctgaaggag aagatcgccaccgccaaggc cgaaggcgcc accctgctgc tgggtggcga 3420 gccccagggc aacgtcatgccgccccatgt gttcggcaac gtcaccgccg acatggaaat 3480 cgcccgcgaa gaaattttcggcccgctggt gggcatccaa tccgcccgtg acgccgaaca 3540 cgccctggag ttggccaacagcagcgagta cggcctgtcc agcgcggtgt tcaccgccag 3600 cctcgagcgc ggcgtgcagttcgcccggcg catccacgcc ggcatgaccc acgtgaacga 3660 catcccggtt aacgacgagcccaacgctcc cttcggcggc gagaagaact ctggcctcgg 3720 ccgcttcaac ggcgactgggccatcgagga gttcaccacc gatcactgga tcaccctgca 3780 acacagcccg cggccctatccgttctgatg ctgccgcatc cccatcaccc agccccaata 3840 aaaaacggag tacgaaatgtcctcactcct caacagccga gctgtgaaac ggccactgct 3900 ggccagcctt gcactaattttcgccctgct cgccggccag gccttcgccg acggcgacgg 3960 cgtctggaaa ggcggcgagaacgtctacca gaaaatctgt ggccactgcc acgaaaaaca 4020 ggtgggcccg gtgatcaccggccgccagct accgccgcag tacatcagtg ccgtggtgcg 4080 caacggcttc cgcgccatgccggcctttcc ggcctcgttc atcgacgaca aggccctgca 4140 gcaggtcgcc gagtacatctccaagacccc tgctactgtg gccaagccct gaggtgccgg 4200 cgatgaacat cgaacgtcgtaccctgctca agggcatggc cctgggcggc ctggctggcg 4260 ccgccatggg cgccttcggcctggcgatga ccaaggccat gctgggcggg caggcccagc 4320 cactgcccac cctcgtcctggtagatggcg aggcggccgg agcggccttc ctcgccggag 4380 tcggttccag cccggcggccagcaaggccg aggtgcagcg caccgatctc ggcctggact 4440 tcgtcttggg cctggagaagcgcctgcgca gtggtcagca gcaacgcatc atcggtctgg 4500 tggatgacgc cagcgccgctctgatcctcg acctggcccg cagcagcggc gcgcgggtgc 4560 agtggctcgg ccagcatagcgccgcggccg gctcctcccg gcaccgtctg ctcagcgccg 4620 acagcgccca gggctgctcccttcgcctgg gccagcagct ccatgcctgc ggcggcggct 4680 tcagcctgag cgaacagcaccccctgggtg gccagcccct gaatctggcc ggtgccgcgc 4740 gcagcggcgg ctccgcgcaatgggcggcca gcatcggcca cgacctggcc agcctgggcg 4800 gcgatgacag cagtgcggccccacgcattg ccaaccatta cccggcgctt accggccaat 4860 tcgtttcgtt ctcgatcctggtttgaagga gctgacagat gaccgagcaa acccagaaca 4920 ccctgattcc ccgtggcgtgaatgacgcca acctccagca agccctggcc aagttccgca 4980 agctgctggg cgaggacaacgtcctggtca aggacgagca actcatcccc tacaacaaga 5040 tcatgatcgc agtggacaacgccgaacacg cgccctccgc tgctgtcacc gccaccactg 5100 tggaacaggt gcagggcgtggtgaagatct gcaacgaata cggcattccg gtgtggacca 5160 tctccaccgg ccgcaacttcggttacggct cggcggcccc cggccagcgt ggccaggtga 5220 tcctcgacct gaagaaaatgaacaagatca tccacgtaga cccggacctg tgcaccgccc 5280 tggtggaacc gggggtgacctaccagcagc tgtacgatta cctggaagag aacaacatcc 5340 cgctgatgct gtccttctctgcaccctcgg ccatcgccgg cccgctgggc aacaccatgg 5400 accgtggcgt gggctacaccccctacggcg agcacttcct catgcagtgc ggcatggaag 5460 tggtgctggc caatggcgacgtctaccgca ccggcatggg cggggtgaaa ggcgacaacg 5520 cctggcaggt gttcaagtggggctacggcc cgaccctgga cggcatgttc acccaggcca 5580 actacggcat ctgcaccaagatgggtttct ggctgatgcc caagcccccg gtgttcaagc 5640 ccttcgagat caagttcgagaacgagtccg acatcagcga gatcgtcgaa ttcatccgtc 5700 cgctgcgcat cgcccaggtcatcccaaact ccgtggtgat cgccggtgtg ctctgggagg 5760 cctccacctg caatacccgccgctcggact acaccactga gccgggcgcc actcccgaca 5820 ccatcctgaa gcagatccagaaggacaagg aactcggcgc ctggaacgtc tatgccgctc 5880 tctacggcac gcaggaacaggtggacgtga actggaagat cgtcaccggc gccctggcca 5940 aactgggcaa gggcaggattgtcacccagg aagaggccgg cgatacccag cccttcaagt 6000 accgttccca gttgatgtccggcgtcccca acctgcagga attcggcctg tacaactggc 6060 gcgggggcgg cggctccatgtggttcgccc cggtcagcca ggcccgtggc atcgagtgcg 6120 acaagcagca ggcgctggccaagaagatcc tcaacaagca cggcctggac tacgtcggcg 6180 agttcattgt cggctggcgcgacatgcacc acgtaatcga cgtgctgtac gaccgcacca 6240 accccgagga aacccaacgcgcctacgcct gcttccacga gttgctggat gagttcgaga 6300 agcacggcta tgcggtgtaccgcgtgaaca ctgcgttcca ggagcgcgtg gcgcagaggt 6360 acggcacggt caagcgcaggtggaacacgc catcaagcgc gccctggacc cgaacaacat 6420 cctggcaccc ggcaaatccggcatcgacct cgccaacaag ttctaaccct aagcaagacc 6480 ccgccgggta a 6491 2 611PRT Pseudomonas mendocina KR-1 2 Met Thr Met Thr Arg Lys Pro Ser Ser AsnPro Gly Arg Ser Met Gly 1 5 10 15 Asp Glu Phe Leu Lys Gln Leu Leu GlyArg Gln Lys Ser Leu Ile Val 20 25 30 Asp Gln Ala Asn Gln Phe Gly Ser SerGly Leu Pro Ser Ala Glu Gln 35 40 45 Leu Ala Glu Thr Val Ala Phe Ala ProGln Asp Gly Asn Ile Trp Leu 50 55 60 Cys Gly Gln Arg Met Met Leu Leu GlnGly Ala Ala Phe Gly Ala Ile 65 70 75 80 Arg Arg Glu Leu Val Glu Ala LeuGly Leu Asp Lys Ala Arg Gly Leu 85 90 95 Leu Thr Arg Ile Gly Trp Gln AlaGly Thr Arg Asp Ala Ala Gln Val 100 105 110 Ser Ala Gln Trp Pro Glu GlyAsp His Ala Ser Leu Tyr Ser Ala Gly 115 120 125 Pro Arg Leu His Met LeuGlu Gly Met Val Asn Val Glu Val Val Arg 130 135 140 Phe Asp Ile Asp SerSer Ile Gly His Phe Tyr Ser Glu Phe Phe Trp 145 150 155 160 His Asn SerLeu Glu Ala Asp Glu His Ile Ala Ser Tyr Gly Leu Gly 165 170 175 Ser GluPro Ala Cys Trp Met Glu Val Gly Tyr Ala Ser Gly Tyr Thr 180 185 190 SerSer Leu Leu Gly Arg Leu Val Val Phe Arg Glu Gln Glu Cys Arg 195 200 205Cys Met Gly His Ser Ala Cys Arg Ile Val Gly Lys Pro Ala Glu Gln 210 215220 Trp Glu Asp Ile Asp Thr Asp Leu Ala His Leu Asp Ala Ser Asp Phe 225230 235 240 Leu Ser Arg Ser Thr Tyr Gly Ser Ala Asp Glu Thr Glu Met AlaAsp 245 250 255 Leu Asp Glu Pro Pro Glu Asp Gln Ala Met Val Gly Ile SerAla Ala 260 265 270 Phe Val Ala Ala Ser Gln Gln Leu Tyr Arg Val Ala SerThr Gln Ala 275 280 285 Thr Val Leu Leu Thr Gly Glu Ser Gly Val Gly LysGlu Leu Phe Ala 290 295 300 Arg Thr Leu His Gln Ala Ser Pro Arg Arg GlnThr Pro Leu Val Ala 305 310 315 320 Leu Asn Cys Ala Ala Leu Pro Glu ThrLeu Leu Glu Ala Glu Leu Phe 325 330 335 Gly Val Glu Arg Gly Ala Phe ThrGly Ala Asp Arg Ser Arg Pro Gly 340 345 350 Arg Phe Glu Arg Ala Lys GlyGly Thr Leu Phe Leu Asp Glu Ile Ala 355 360 365 Thr Leu Ser Leu Ser AlaGln Ser Lys Ile Leu Arg Val Leu Gln Glu 370 375 380 Gly Glu Ile Glu ArgVal Gly Gly Thr Ser Ala Ile Pro Val Asp Val 385 390 395 400 Arg Val IleAla Ala Thr Asn Leu Asp Leu Arg Arg Glu Val Glu Ala 405 410 415 Gly ArgPhe Arg Glu Asp Leu Leu Tyr Arg Leu Asn Val Phe Pro Ile 420 425 430 HisLeu Pro Pro Leu Arg Glu Arg Arg Glu Asp Ile Pro Leu Leu Met 435 440 445Ser Tyr Phe Leu Arg Lys Phe Ser Ala Arg His Gly Leu Lys Pro Leu 450 455460 Gly Phe Ser Thr Arg Leu Val Asn Ala Leu Leu Thr Tyr Arg Phe Pro 465470 475 480 Gly Asn Ile Arg Glu Leu Gln Asn Leu Ile Glu Arg Gly Val IleAla 485 490 495 Ala Gly Ser Gly Glu Leu Ile Asp Leu Val His Leu Ala LysAla Gly 500 505 510 Glu Pro Leu Arg Ser Ser Gly Ile Gly Leu Thr Ala GluGly Arg Leu 515 520 525 Asp Ala Val Gly Gly Glu Pro Gln Ala Arg Ala GluVal Val Val Glu 530 535 540 Glu His Glu Pro Val Glu Leu Gly Leu Ala AsnLeu Gln Ala Phe Leu 545 550 555 560 Ala Gly Arg Asp Arg Val Leu Gly ThrSer Leu Lys Glu Ile Glu Gln 565 570 575 Arg Leu Val His Leu Ala Leu GluArg Thr Gly Gly Asn Leu Thr Ala 580 585 590 Ala Ala Gln Met Leu Gly MetSer Arg Ala Gln Ile Ser Tyr Arg Leu 595 600 605 Lys Gly Glu 610 3 491PRT Pseudomonas mendocina KR-1 3 Met Asp Thr Thr Arg Pro Ala Tyr Gln AsnLeu Glu Leu Gln Pro Leu 1 5 10 15 Ala Gly Gln Trp Arg Ala Gly Ser SerGly Arg Pro Leu Glu Val Phe 20 25 30 Asp Pro Tyr Asn Asp Glu Leu Leu LeuArg Ile Ala Leu Ala Ser Arg 35 40 45 Glu Asp Leu Asp Ala Ala Tyr Arg LysAla Arg Asp Ser Gln Arg Glu 50 55 60 Trp Ala Thr Thr Ala Pro Ala Glu ArgAla Arg Val Leu Leu Glu Ala 65 70 75 80 Val Lys Ile Phe Asp Glu Arg ArgGlu Glu Ile Ile Asp Trp Ile Ile 85 90 95 Arg Glu Ser Gly Ser Thr Arg IleLys Ala Gln Ile Glu Trp Gly Ala 100 105 110 Ala Arg Ala Ile Thr Leu GluSer Ala Ser Leu Pro Asn Arg Val His 115 120 125 Gly Arg Ile Ile Ala SerAsn Ile Ser Gly Lys Glu Ser Arg Val Tyr 130 135 140 Arg Ala Pro Leu GlyVal Ile Gly Val Ile Ser Pro Trp Asn Phe Pro 145 150 155 160 Leu His LeuThr Ala Arg Ser Leu Ala Pro Ala Leu Ala Leu Gly Asn 165 170 175 Ala ValVal Val Lys Pro Ala Ser Asp Thr Pro Ile Thr Gly Gly Leu 180 185 190 LeuLeu Ala Arg Ile Phe Glu Glu Ala Gly Leu Pro Ala Gly Val Leu 195 200 205Ser Val Val Val Gly Ser Gly Ala Glu Ile Gly Asp Ala Phe Val Glu 210 215220 His Pro Val Pro Ala Leu Ile Ser Phe Thr Gly Ser Thr Gln Val Gly 225230 235 240 Arg Asn Ile Gly Arg Ile Ala Ser Gly Gly Glu His Leu Lys HisVal 245 250 255 Ala Leu Glu Leu Gly Gly Asn Ser Pro Phe Val Val Leu AlaAsp Ala 260 265 270 Asp Val Glu Gln Ala Val Asn Ala Ala Val Val Gly LysPhe Leu His 275 280 285 Gln Gly Gln Ile Cys Met Ala Ile Asn Arg Ile IleVal Glu Gln Pro 290 295 300 Leu Leu Glu Asp Phe Thr Arg Arg Phe Val GluArg Val Lys Ala Leu 305 310 315 320 Pro Tyr Gly Asp Pro Ser Lys Pro GlyThr Val Val Gly Pro Val Ile 325 330 335 Asn Ala Arg Gln Leu Ala Gly LeuLys Glu Lys Ile Ala Thr Ala Lys 340 345 350 Ala Glu Gly Ala Thr Leu LeuLeu Gly Gly Glu Pro Gln Gly Asn Val 355 360 365 Met Pro Pro His Val PheGly Asn Val Thr Ala Asp Met Glu Ile Ala 370 375 380 Arg Glu Glu Ile PheGly Pro Leu Val Gly Ile Gln Ser Ala Arg Asp 385 390 395 400 Ala Glu HisAla Leu Glu Leu Ala Asn Ser Ser Glu Tyr Gly Leu Ser 405 410 415 Ser AlaVal Phe Thr Ala Ser Leu Glu Arg Gly Val Gln Phe Ala Arg 420 425 430 ArgIle His Ala Gly Met Thr His Val Asn Asp Ile Pro Val Asn Asp 435 440 445Glu Pro Asn Ala Pro Phe Gly Gly Glu Lys Asn Ser Gly Leu Gly Arg 450 455460 Phe Asn Gly Asp Trp Ala Ile Glu Glu Phe Thr Thr Asp His Trp Ile 465470 475 480 Thr Leu Gln His Ser Pro Arg Pro Tyr Pro Phe 485 490 4 111PRT Pseudomonas mendocina KR-1 4 Met Ser Ser Leu Leu Asn Ser Arg Ala ValLys Arg Pro Leu Leu Ala 1 5 10 15 Ser Leu Ala Leu Ile Phe Ala Leu LeuAla Gly Gln Ala Phe Ala Asp 20 25 30 Gly Asp Gly Val Trp Lys Gly Gly GluAsn Val Tyr Gln Lys Ile Cys 35 40 45 Gly His Cys His Glu Lys Gln Val GlyPro Val Ile Thr Gly Arg Gln 50 55 60 Leu Pro Pro Gln Tyr Ile Ser Ala ValVal Arg Asn Gly Phe Arg Ala 65 70 75 80 Met Pro Ala Phe Pro Ala Ser PheIle Asp Asp Lys Ala Leu Gln Gln 85 90 95 Val Ala Glu Tyr Ile Ser Lys ThrPro Ala Thr Val Ala Lys Pro 100 105 110 5 227 PRT Pseudomonas mendocinaKR-1 5 Met Asn Ile Glu Arg Arg Thr Leu Leu Lys Gly Met Ala Leu Gly Gly 15 10 15 Leu Ala Gly Ala Ala Met Gly Ala Phe Gly Leu Ala Met Thr Lys Ala20 25 30 Met Leu Gly Gly Gln Ala Gln Pro Leu Pro Thr Leu Val Leu Val Asp35 40 45 Gly Glu Ala Ala Gly Ala Ala Phe Leu Ala Gly Val Gly Ser Ser Pro50 55 60 Ala Ala Ser Lys Ala Glu Val Gln Arg Thr Asp Leu Gly Leu Asp Phe65 70 75 80 Val Leu Gly Leu Glu Lys Arg Leu Arg Ser Gly Gln Gln Gln ArgIle 85 90 95 Ile Gly Leu Val Asp Asp Ala Ser Ala Ala Leu Ile Leu Asp LeuAla 100 105 110 Arg Ser Ser Gly Ala Arg Val Gln Trp Leu Gly Gln His SerAla Ala 115 120 125 Ala Gly Ser Ser Arg His Arg Leu Leu Ser Ala Asp SerAla Gln Gly 130 135 140 Cys Ser Leu Arg Leu Gly Gln Gln Leu His Ala CysGly Gly Gly Phe 145 150 155 160 Ser Leu Ser Glu Gln His Pro Leu Gly GlyGln Pro Leu Asn Leu Ala 165 170 175 Gly Ala Ala Arg Ser Gly Gly Ser AlaGln Trp Ala Ala Ser Ile Gly 180 185 190 His Asp Leu Ala Ser Leu Gly GlyAsp Asp Ser Ser Ala Ala Pro Arg 195 200 205 Ile Ala Asn His Tyr Pro AlaLeu Thr Gly Gln Phe Val Ser Phe Ser 210 215 220 Ile Leu Val 225 6 530PRT Pseudomonas mendocina KR-1 6 Met Thr Glu Gln Thr Gln Asn Thr Leu IlePro Arg Gly Val Asn Asp 1 5 10 15 Ala Asn Leu Gln Gln Ala Leu Ala LysPhe Arg Lys Leu Leu Gly Glu 20 25 30 Asp Asn Val Leu Val Lys Asp Glu GlnLeu Ile Pro Tyr Asn Lys Ile 35 40 45 Met Ile Ala Val Asp Asn Ala Glu HisAla Pro Ser Ala Ala Val Thr 50 55 60 Ala Thr Thr Val Glu Gln Val Gln GlyVal Val Lys Ile Cys Asn Glu 65 70 75 80 Tyr Gly Ile Pro Val Trp Thr IleSer Thr Gly Arg Asn Phe Gly Tyr 85 90 95 Gly Ser Ala Ala Pro Gly Gln ArgGly Gln Val Ile Leu Asp Leu Lys 100 105 110 Lys Met Asn Lys Ile Ile HisVal Asp Pro Asp Leu Cys Thr Ala Leu 115 120 125 Val Glu Pro Gly Val ThrTyr Gln Gln Leu Tyr Asp Tyr Leu Glu Glu 130 135 140 Asn Asn Ile Pro LeuMet Leu Ser Phe Ser Ala Pro Ser Ala Ile Ala 145 150 155 160 Gly Pro LeuGly Asn Thr Met Asp Arg Gly Val Gly Tyr Thr Pro Tyr 165 170 175 Gly GluHis Phe Leu Met Gln Cys Gly Met Glu Val Val Leu Ala Asn 180 185 190 GlyAsp Val Tyr Arg Thr Gly Met Gly Gly Val Lys Gly Asp Asn Ala 195 200 205Trp Gln Val Phe Lys Trp Gly Tyr Gly Pro Thr Leu Asp Gly Met Phe 210 215220 Thr Gln Ala Asn Tyr Gly Ile Cys Thr Lys Met Gly Phe Trp Leu Met 225230 235 240 Pro Lys Pro Pro Val Phe Lys Pro Phe Glu Ile Lys Phe Glu AsnGlu 245 250 255 Ser Asp Ile Ser Glu Ile Val Glu Phe Ile Arg Pro Leu ArgIle Ala 260 265 270 Gln Val Ile Pro Asn Ser Val Val Ile Ala Gly Val LeuTrp Glu Ala 275 280 285 Ser Thr Cys Asn Thr Arg Arg Ser Asp Tyr Thr ThrGlu Pro Gly Ala 290 295 300 Thr Pro Asp Thr Ile Leu Lys Gln Ile Gln LysAsp Lys Glu Leu Gly 305 310 315 320 Ala Trp Asn Val Tyr Ala Ala Leu TyrGly Thr Gln Glu Gln Val Asp 325 330 335 Val Asn Trp Lys Ile Val Thr GlyAla Leu Ala Lys Leu Gly Lys Gly 340 345 350 Arg Ile Val Thr Gln Glu GluAla Gly Asp Thr Gln Pro Phe Lys Tyr 355 360 365 Arg Ser Gln Leu Met SerGly Val Pro Asn Leu Gln Glu Phe Gly Leu 370 375 380 Tyr Asn Trp Arg GlyGly Gly Gly Ser Met Trp Phe Ala Pro Val Ser 385 390 395 400 Gln Ala ArgGly Ile Glu Cys Asp Lys Gln Gln Ala Leu Ala Lys Lys 405 410 415 Ile LeuAsn Lys His Gly Leu Asp Tyr Val Gly Glu Phe Ile Val Gly 420 425 430 TrpArg Asp Met His His Val Ile Asp Val Leu Tyr Asp Arg Thr Asn 435 440 445Pro Glu Glu Thr Gln Arg Ala Tyr Ala Cys Phe His Glu Leu Leu Asp 450 455460 Glu Phe Glu Lys His Gly Tyr Ala Val Tyr Arg Val Asn Thr Ala Phe 465470 475 480 Gln Glu Arg Val Ala Gln Arg Tyr Gly Thr Val Lys Arg Arg TrpAsn 485 490 495 Thr Pro Ser Ser Ala Pro Trp Thr Arg Thr Thr Ser Trp HisPro Ala 500 505 510 Asn Pro Ala Ser Thr Ser Pro Thr Ser Ser Asn Pro LysGln Asp Pro 515 520 525 Ala Gly 530 7 20 DNA Artificial SequenceDescription of Artificial Sequence primer 7 atgaccatga ctcgcaaacc 20 820 DNA Artificial Sequence Description of Artificial Sequence primer 8tttgcgagtc atggtcatgg 20 9 20 DNA Artificial Sequence Description ofArtificial Sequence primer 9 cgcgcaaaca agagagaacc 20 10 22 DNAArtificial Sequence Description of Artificial Sequence primer 10cattcgatct gcgccttgat gc 22 11 20 DNA Artificial Sequence Description ofArtificial Sequence primer 11 tttgtggtct tggccgatgc 20 12 20 DNAArtificial Sequence Description of Artificial Sequence primer 12tgacgttgcc gaacacatgg 20 13 21 DNA Artificial Sequence Description ofArtificial Sequence primer 13 cccagcccca ataaaaaacg g 21 14 24 DNAArtificial Sequence Description of Artificial Sequence primer 14gattttctgg tagacgttct cgcc 24 15 21 DNA Artificial Sequence Descriptionof Artificial Sequence primer 15 cgtctaccag aaaatctgtg g 21 16 20 DNAArtificial Sequence Description of Artificial Sequence primer 16atgctgtcgt tctctgcacc 20 17 21 DNA Artificial Sequence Description ofArtificial Sequence primer 17 gacacatcct gaagcagatc c 21 18 21 DNAArtificial Sequence Description of Artificial Sequence primer 18tgaacactgc gttccaggag c 21 19 21 DNA Artificial Sequence Description ofArtificial Sequence primer 19 ggcagaagag cctgatcgtc g 21 20 19 DNAArtificial Sequence Description of Artificial Sequence primer 20actccggaga agtttctgc 19 21 19 DNA Artificial Sequence Description ofArtificial Sequence primer 21 atcagtccgt ggaacttcc 19 22 18 DNAArtificial Sequence Description of Artificial Sequence primer 22ttaacgacga gcccaacg 18 23 20 DNA Artificial Sequence Description ofArtificial Sequence primer 23 tgttcggcgt tgtccactgc 20 24 18 DNAArtificial Sequence Description of Artificial Sequence primer 24aacttcggtt acggctcg 18 25 18 DNA Artificial Sequence Description ofArtificial Sequence primer 25 gctcggacta caccactg 18 26 20 DNAArtificial Sequence Description of Artificial Sequence primer 26atggcaatat ctggctctgc 20 27 18 DNA Artificial Sequence Description ofArtificial Sequence primer 27 aaaccctcga gcaatccc 18 28 19 DNAArtificial Sequence Description of Artificial Sequence primer 28atgaccaagg ccatgctgg 19 29 18 DNA Artificial Sequence Description ofArtificial Sequence primer 29 ttcgctcagg ctgaaacc 18 30 21 DNAArtificial Sequence Description of Artificial Sequence primer 30gtaatggttg gcaatgcgtg g 21 31 19 DNA Artificial Sequence Description ofArtificial Sequence primer 31 tggatggaag tgggctacg 19 32 22 DNAArtificial Sequence Description of Artificial Sequence primer 32ttcgacatcg actccagcat cg 22 33 18 DNA Artificial Sequence Description ofArtificial Sequence primer 33 agctgctatt gcgcatcg 18 34 18 DNAArtificial Sequence Description of Artificial Sequence primer 34ttggtggcgc tcaactgc 18 35 21 DNA Artificial Sequence Description ofArtificial Sequence primer 35 aaatggccga tctggatgag c 21 36 22 DNAArtificial Sequence Description of Artificial Sequence primer 36tatcccactc cactgtccat gg 22 37 22 DNA Artificial Sequence Description ofArtificial Sequence primer 37 caccacttcc atgccgcact gc 22 38 21 DNAArtificial Sequence Description of Artificial Sequence primer 38tgcggtaaag ctctccattg g 21 39 22 DNA Artificial Sequence Description ofArtificial Sequence primer 39 caagtaatcg tacagctgct gg 22 40 19 DNAArtificial Sequence Description of Artificial Sequence primer 40agccgtaccc gaagttgcg 19 41 24 DNA Artificial Sequence Description ofArtificial Sequence primer 41 tgcgatcatg atcttgttgt aggg 24 42 24 DNAArtificial Sequence Description of Artificial Sequence primer 42gatcgagaac gaaacgaatt ggcc 24 43 24 DNA Artificial Sequence Descriptionof Artificial Sequence primer 43 gtgctgttcg ctcaagctga aacc 24 44 24 DNAArtificial Sequence Description of Artificial Sequence primer 44accacaccga tgatgccttg ctgc 24 45 23 DNA Artificial Sequence Descriptionof Artificial Sequence primer 45 tatgctggcc gatccactgc acc 23 46 23 DNAArtificial Sequence Description of Artificial Sequence primer 46ccttgagcag ggtacgacgt tcg 23 47 24 DNA Artificial Sequence Descriptionof Artificial Sequence primer 47 atcaccgggc ccacctgttt ttcg 24 48 24 DNAArtificial Sequence Description of Artificial Sequence primer 48ttggagatgt actcggcgac ctgc 24 49 24 DNA Artificial Sequence Descriptionof Artificial Sequence primer 49 tgttgcaggg tgatccagtg atcg 24 50 24 DNAArtificial Sequence Description of Artificial Sequence primer 50actccgtttt ttattggggc tggg 24 51 22 DNA Artificial Sequence Descriptionof Artificial Sequence primer 51 tggcgatctt ctccttcaaa cc 22 52 18 DNAArtificial Sequence Description of Artificial Sequence primer 52gccgaaaatt tcttcgcg 18 53 17 DNA Artificial Sequence Description ofArtificial Sequence primer 53 tggtgcagga acttgcc 17 54 19 DNA ArtificialSequence Description of Artificial Sequence primer 54 gccttgatacgggtgctgc 19 55 23 DNA Artificial Sequence Description of ArtificialSequence primer 55 aagttccacg gactgatcac gcc 23 56 24 DNA ArtificialSequence Description of Artificial Sequence primer 56 attcgatctgcgccttgatg cggg 24 57 24 DNA Artificial Sequence Description ofArtificial Sequence primer 57 gtccatgggt tctctcttgt ttgc 24 58 24 DNAArtificial Sequence Description of Artificial Sequence primer 58ctgtccttta acaactaacg tgcc 24 59 18 DNA Artificial Sequence Descriptionof Artificial Sequence primer 59 gatacgtcag gcaagacg 18 60 24 DNAArtificial Sequence Description of Artificial Sequence primer 60ctgaatagca ttgttcctac ctcc 24 61 28 DNA Artificial Sequence Descriptionof Artificial Sequence primer 61 caccaagctt tgttgatctc ccttcaag 28 62 27DNA Artificial Sequence Description of Artificial Sequence primer 62ttcggcgcca tccgtcgcga gttggtg 27 63 18 DNA Artificial SequenceDescription of Artificial Sequence primer 63 agcggcatcg gcctgacc 18 6422 DNA Artificial Sequence Description of Artificial Sequence primer 64gttgtcaaag aacatgaacc gg 22 65 24 DNA Artificial Sequence Description ofArtificial Sequence primer 65 tatgtctggc cctctgtgcg gttg 24 66 22 DNAArtificial Sequence Description of Artificial Sequence primer 66agtatgtctc tggccctcgg tg 22 67 20 DNA Artificial Sequence Description ofArtificial Sequence primer 67 taaacatgcc cagacggtgg 20 68 21 DNAArtificial Sequence Description of Artificial Sequence primer 68gatttgcaga accgtctgtc c 21 69 23 DNA Artificial Sequence Description ofArtificial Sequence primer 69 tacggcatgt gcaccaagat ggg 23 70 24 DNAArtificial Sequence Description of Artificial Sequence primer 70aggaattcgg cctgtactac tggc 24 71 24 DNA Artificial Sequence Descriptionof Artificial Sequence primer 71 agttgctgga tgagttcgag aagc 24 72 27 DNAArtificial Sequence Description of Artificial Sequence primer 72gcatgatgga tcctgcacgt gatatgg 27 73 19 DNA Artificial SequenceDescription of Artificial Sequence primer 73 tgggaacggt acttgaagg 19 7417 DNA Artificial Sequence Description of Artificial Sequence primer 74gttttcccag tcacgac 17 75 24 DNA Artificial Sequence Description ofArtificial Sequence primer 75 agcggataac aatttcacac agga 24 76 17 DNAArtificial Sequence Description of Artificial Sequence primer 76gtaaaacgac ggccagt 17 77 16 DNA Artificial Sequence Description ofArtificial Sequence primer 77 aacagctatg accatg 16 78 23 DNA ArtificialSequence Description of Artificial Sequence primer 78 gaaattggagctccaaatga cat 23 79 23 DNA Artificial Sequence Description ofArtificial Sequence primer 79 ctcatgacag gatcctcaag gct 23 80 23 DNAArtificial Sequence Description of Artificial Sequence primer 80gttcatacca gtctttacgt ggg 23 81 22 DNA Artificial Sequence Descriptionof Artificial Sequence primer 81 ttccatactc tgtacccagc cc 22 82 21 DNAArtificial Sequence Description of Artificial Sequence primer 82attccggtct gcatcaactg c 21 83 18 DNA Artificial Sequence Description ofArtificial Sequence primer 83 tggtggtatt cggtaccg 18 84 20 DNAArtificial Sequence Description of Artificial Sequence primer 84tacttccata ctctgtaccc 20 85 22 DNA Artificial Sequence Description ofArtificial Sequence primer 85 agcaccgaaa cgccagtcat cc 22 86 20 DNAArtificial Sequence Description of Artificial Sequence primer 86gcggacatcc atagagaagc 20 87 18 DNA Artificial Sequence Description ofArtificial Sequence primer 87 atctctaata ccggtgcc 18 88 20 DNAArtificial Sequence Description of Artificial Sequence primer 88aagcataacc gctcaaaggc 20 89 24 DNA Artificial Sequence Description ofArtificial Sequence primer 89 attgccccca cgattattgc gacc 24 90 19 DNAArtificial Sequence Description of Artificial Sequence primer 90gattgcccca taaccctcc 19 91 1562 DNA Pseudomonas mendocina KR-1 91ggtagttttc ttcaggattt ctctaaacta tcgtttatca aacgataaac cttggttcgc 60ttaattgcga aaattgcata aaccaataat ccaaaaaaca atttattttt atttcgtggt 120cgcaataatc gtgggtgcaa tcaaacggta ttttcctgct tcactttata agaataagaa 180gaggtagaaa gatgataaaa atgaaaattg ccagcgtact cgtactgcct ttgagcggtt 240atgcttttag cgtgcacgct acacaggtgt tcgatctgga gggttatggg gcaatctctc 300gtgccatggg aggtaccagc tcatcgtatt ataccggcaa tgctgcattg atcagcaacc 360ccgctacatt gagcttggct ccggacggaa gtcagtttga gctcgggccg gatatagtaa 420gtaccgatat tgaggttcgt gacagcagcg gtgcgaaagt aaaaagcagc acggaatcca 480ataatcgagg cccctatatc ggtccgcagt tgagctatgt tactcagctg gatgactggc 540gtttcggtgc tgggttgttt gtgagtagtg ggctgggtac agagtatgga agtaacagtt 600tcttgtcaca gacagaaaat ggcacccaaa ccagctttga caattccagc cgtctgattg 660tgttgcgcgc tcctgtaggc tttagttatc aagtaacacc acaacttaca gtcggcgcaa 720gtgctgatct ggtctggacc tcactcaatc tcgagcttct actcccatca tctcaggtgg 780gagcactcgc tgcgcagggt aatctttcag gtgatttagt cgccccactc gctgggtttg 840tgggtgctgg tggtgctgca catttcagtc taagtcgcaa caacccagtt ggcggtgccg 900tggatgcaat cgggtggggt gggcgtttgg gtctgaccta caagctcacg gataagacag 960tccttggtgc gatgtacaac ttcaagactt ctgtgggcga cctcgaaggg acggcaacac 1020tttctgctat cagcggtgat ggtgcggtgt tgccattaca tggcgatatc cgcgtaaaag 1080acttcgagat gcccgccagt ctgacgttcg gctttgctca tcaattcaac gagcgttggc 1140tggttgctgc tgatgtcaag cgtgtctact ggagcgatgt catggaagac atcagtgtgg 1200atttcaaatc gcagtcaggt gggattgata tcgaattacc acacaactat caggatatta 1260cggtggcctc catcggcacc gcttacagag ttaatgacaa gctaactctt cgtgctggat 1320atagctatgc gcaacaggcg ctggacagta ggctgatatt gccagtaatt ccagcttatt 1380tgaagaaaca cgtttctctc ggtagcgatt atagttttga taaaaaatca aaactcaatt 1440tggcgatttc ttttggccta aaagagagct tgaacacacc atcataccta agcggcaccg 1500aaacgttgaa gcaaagccac agccaaataa acgcagtggt ttcctacagc aaaagctttt 1560aa 1562 92 456 PRT Pseudomonas mendocina KR-1 92 Met Ile Lys Met Lys IleAla Ser Val Leu Val Leu Pro Leu Ser Gly 1 5 10 15 Tyr Ala Phe Ser ValHis Ala Thr Gln Val Phe Asp Leu Glu Gly Tyr 20 25 30 Gly Ala Ile Ser ArgAla Met Gly Gly Thr Ser Ser Ser Tyr Tyr Thr 35 40 45 Gly Asn Ala Ala LeuIle Ser Asn Pro Ala Thr Leu Ser Leu Ala Pro 50 55 60 Asp Gly Ser Gln PheGlu Leu Gly Pro Asp Ile Val Ser Thr Asp Ile 65 70 75 80 Glu Val Arg AspSer Ser Gly Ala Lys Val Lys Ser Ser Thr Glu Ser 85 90 95 Asn Asn Arg GlyPro Tyr Ile Gly Pro Gln Leu Ser Tyr Val Thr Gln 100 105 110 Leu Asp AspTrp Arg Phe Gly Ala Gly Leu Phe Val Ser Ser Gly Leu 115 120 125 Gly ThrGlu Tyr Gly Ser Asn Ser Phe Leu Ser Gln Thr Glu Asn Gly 130 135 140 ThrGln Thr Ser Phe Asp Asn Ser Ser Arg Leu Ile Val Leu Arg Ala 145 150 155160 Pro Val Gly Phe Ser Tyr Gln Val Thr Pro Gln Leu Thr Val Gly Ala 165170 175 Ser Ala Asp Leu Val Trp Thr Ser Leu Asn Leu Glu Leu Leu Leu Pro180 185 190 Ser Ser Gln Val Gly Ala Leu Ala Ala Gln Gly Asn Leu Ser GlyAsp 195 200 205 Leu Val Ala Pro Leu Ala Gly Phe Val Gly Ala Gly Gly AlaAla His 210 215 220 Phe Ser Leu Ser Arg Asn Asn Pro Val Gly Gly Ala ValAsp Ala Ile 225 230 235 240 Gly Trp Gly Gly Arg Leu Gly Leu Thr Tyr LysLeu Thr Asp Lys Thr 245 250 255 Val Leu Gly Ala Met Tyr Asn Phe Lys ThrSer Val Gly Asp Leu Glu 260 265 270 Gly Thr Ala Thr Leu Ser Ala Ile SerGly Asp Gly Ala Val Leu Pro 275 280 285 Leu His Gly Asp Ile Arg Val LysAsp Phe Glu Met Pro Ala Ser Leu 290 295 300 Thr Phe Gly Phe Ala His GlnPhe Asn Glu Arg Trp Leu Val Ala Ala 305 310 315 320 Asp Val Lys Arg ValTyr Trp Ser Asp Val Met Glu Asp Ile Ser Val 325 330 335 Asp Phe Lys SerGln Ser Gly Gly Ile Asp Ile Glu Leu Pro His Asn 340 345 350 Tyr Gln AspIle Thr Val Ala Ser Ile Gly Thr Ala Tyr Arg Val Asn 355 360 365 Asp LysLeu Thr Leu Arg Ala Gly Tyr Ser Tyr Ala Gln Gln Ala Leu 370 375 380 AspSer Arg Leu Ile Leu Pro Val Ile Pro Ala Tyr Leu Lys Lys His 385 390 395400 Val Ser Leu Gly Ser Asp Tyr Ser Phe Asp Lys Lys Ser Lys Leu Asn 405410 415 Leu Ala Ile Ser Phe Gly Leu Lys Glu Ser Leu Asn Thr Pro Ser Tyr420 425 430 Leu Ser Gly Thr Glu Thr Leu Lys Gln Ser His Ser Gln Ile AsnAla 435 440 445 Val Val Ser Tyr Ser Lys Ser Phe 450 455 93 26 DNAArtificial Sequence Description of Artificial Sequence primer 93gatgatgaag cttccccacc aaaccc 26 94 30 DNA Artificial SequenceDescription of Artificial Sequence primer 94 tcatagatca agcttttcccagtcacgacg 30 95 25 DNA Artificial Sequence Description of ArtificialSequence primer 95 ggggatcctc accgccggct caagg 25 96 25 DNA ArtificialSequence Description of Artificial Sequence primer 96 gcgggtgggatccatgggtt ctctc 25 97 23 DNA Artificial Sequence Description ofArtificial Sequence primer 97 cggtacttac tatatccggc ccg 23 98 1836 DNAPseudomonas mendocina KR-1 98 tcactccccc ttgagccggt agctgatctgcgcgcgactc atgcccaaca tctgcgccgc 60 cgcggtgagg ttgccgccgg tgcgttccagggcaaggtgc accaggcgct gctcgatctc 120 cttcagtgat gtgcctagta cccggtcgcgcccggcgagg aaggcctgca ggttggccag 180 ccccagctca accggttcat gctcctcgacaaccacctca gcccgcgctt gcggttcgcc 240 gccgacggca tccagacggc cttcggcggtcaggccgatg ccgctggagc gaagtggctc 300 gccggctttt gccaggtgca ccaggtcgatcagctcgcca ctgcctgcgg cgatcacgcc 360 gcgctcgatc aggttctgca gctcacggatattgccgggg aagcggtagg tcagcagcgc 420 gttgaccagc cgcgtgctga aacccaggggtttgagccca tggcgcgcac tgaacttgcg 480 caggaagtag ctcatcagga gcgggatgtcctcacggcgc tcgcgcaggg gcggcagatg 540 gatggggaac acgttcagcc ggtacagcaggtcctcgcgg aagcgcccgg cctcgacctc 600 tcggcgcagg tccagattgg tggcggcgatcaccctcaca tccaccggga tcgccgaggt 660 accacctacc cgctcgatct cgccctcctgcagcacccgc aggatcttgc tctgggcgct 720 gaggctcagg gtggcgatct cgtcgaggaacagggtgccg cccttggccc gctcgaagcg 780 ccccgggcgg gaacggtcgg cgccggtgaaggcaccgcgc tccacgccga acagttcggc 840 ttccagcaga gtttccggca acgccgcgcagttgagcgcc accaacggcg tttggcggcg 900 cgggctggcc tggtgcaggg tgcgcgcgaagagctccttg cccacccccg attcaccggt 960 cagcagtacg gtggcctggg tcgacgcaacgcggtagagc tgctggctgg cggcgacaaa 1020 ggcggcggaa atgcccacca tggcctggtcctcgggcggc tcatccagat cggccatttc 1080 cgtctcgtct gccgagccgt aggtgctccggctgaggaag tcgctggcat ccaggtgggc 1140 caggtcggtg tcgatgtcct cccactgctccgccggcttg ccgacgatgc ggcacgccga 1200 atggcccatg cagcggcatt cctgctcgcggaacaccacc aggcgcccca gcagggagga 1260 ggtgtagccg ctggcgtagc ccacttccatccagcaggcc ggttcgctgc ccagcccgta 1320 gctggcgatg tgctcgtcgg cttccagggagttgtgccag aagaattcgg aatagaaatg 1380 cccgatgctg gagtcgatgt cgaagcgcaccacttccacg ttcaccatgc cctccagcat 1440 gtgcaggcgc gggcctgcgc tgtagaggctggcgtggtcg ccctcgggcc actgcgcgct 1500 gacctgagcg gcatccctcg ttccggcctgccagccaatg cgggtcagaa ggccacgggc 1560 cttgtcgagg ccgagggctt ccaccaactcgcgacggatg gcgccgaagg cggccccctg 1620 cagcagcatc atgcgctggc cgcagagccagatattgcca tcctggggcg cgaaggcgac 1680 ggtctccgcc agttgctcgg ccgagggcagcccgctgctg ccgaactggt tggcctggtc 1740 gacgatcagg ctcttctgcc ggccgagcaactgcttgagg aattcatccc ccatgctgcg 1800 gccgggattg ctcgagggtt tgcgagtcatggtcat 1836 99 1476 DNA Pseudomonas mendocina KR-1 99 atggacaccacccgccctgc ctaccagaac ctcgagctcc aacctctcgc cgggcaatgg 60 cgcgccggcagtagcggtcg cccgttggag gtcttcgacc cctacaacga cgagctgcta 120 ttgcgcatcgccctggccag ccgcgaagac ctcgacgcag cctaccgcaa ggcccgcgac 180 agccagcgggagtgggcgac cacggcgccg gccgagcgcg cccgggtgct gctggaagcg 240 gtgaagatcttcgatgagcg ccgcgaggag attatcgact ggatcatccg cgagtccggc 300 agcacccgcatcaaggcgca gatcgaatgg ggcgccgccc gcgccatcac cctggagtcg 360 gccagcctgccgaatcgcgt gcacgggcgc atcatcgcct ccaacatctc cggcaaggag 420 agccgcgtgtaccgcgcgcc cctgggcgtg atcggcgtga tcagtccgtg gaacttcccc 480 ctgcacctcactgcccgctc cctggccccg gccctggccc tgggcaatgc cgtggtggtc 540 aagccggccagcgacacccc gatcaccggt ggcctactgc tggcgcgcat cttcgaagaa 600 gccggcctgccggcgggcgt gctcagcgtg gtggtgggtt cgggcgcgga gattggtgac 660 gccttcgtcgagcacccggt gcccgccctc atttccttca ccggctccac tcaggtgggc 720 cgcaacatcggccgcatcgc cagcggcggt gagcacctca agcacgtggc gctggaactg 780 ggcggcaacagcccgtttgt ggtcttggcc gatgccgacg tggagcaggc ggtgaatgcg 840 gccgtggtcggcaagttcct gcaccagggc cagatctgca tggcgatcaa ccgcattatc 900 gtcgagcagcctttgctgga agatttcacc cgccgcttcg tcgagcgcgt caaggccctg 960 ccctatggcgacccgagcaa gccggggacc gtggtcggtc cggtgatcaa cgccaggcag 1020 ctggccggtctgaaggagaa gatcgccacc gccaaggccg aaggcgccac cctgctgctg 1080 ggtggcgagccccagggcaa cgtcatgccg ccccatgtgt tcggcaacgt caccgccgac 1140 atggaaatcgcccgcgaaga aattttcggc ccgctggtgg gcatccaatc cgcccgtgac 1200 gccgaacacgccctggagtt ggccaacagc agcgagtacg gcctgtccag cgcggtgttc 1260 accgccagcctcgagcgcgg cgtgcagttc gcccggcgca tccacgccgg catgacccac 1320 gtgaacgacatcccggttaa cgacgagccc aacgctccct tcggcggcga gaagaactct 1380 ggcctcggccgcttcaacgg cgactgggcc atcgaggagt tcaccaccga tcactggatc 1440 accctgcaacacagcccgcg gccctatccg ttctga 1476 100 336 DNA Pseudomonas mendocina KR-1100 atgtcctcac tcctcaacag ccgagctgtg aaacggccac tgctggccag ccttgcacta 60attttcgccc tgctcgccgg ccaggccttc gccgacggcg acggcgtctg gaaaggcggc 120gagaacgtct accagaaaat ctgtggccac tgccacgaaa aacaggtggg cccggtgatc 180accggccgcc agctaccgcc gcagtacatc agtgccgtgg tgcgcaacgg cttccgcgcc 240atgccggcct ttccggcctc gttcatcgac gacaaggccc tgcagcaggt cgccgagtac 300atctccaaga cccctgctac tgtggccaag ccctga 336 101 684 DNA Pseudomonasmendocina KR-1 101 atgaacatcg aacgtcgtac cctgctcaag ggcatggccctgggcggcct ggctggcgcc 60 gccatgggcg ccttcggcct ggcgatgacc aaggccatgctgggcgggca ggcccagcca 120 ctgcccaccc tcgtcctggt agatggcgag gcggccggagcggccttcct cgccggagtc 180 ggttccagcc cggcggccag caaggccgag gtgcagcgcaccgatctcgg cctggacttc 240 gtcttgggcc tggagaagcg cctgcgcagt ggtcagcagcaacgcatcat cggtctggtg 300 gatgacgcca gcgccgctct gatcctcgac ctggcccgcagcagcggcgc gcgggtgcag 360 tggctcggcc agcatagcgc cgcggccggc tcctcccggcaccgtctgct cagcgccgac 420 agcgcccagg gctgctccct tcgcctgggc cagcagctccatgcctgcgg cggcggcttc 480 agcctgagcg aacagcaccc cctgggtggc cagcccctgaatctggccgg tgccgcgcgc 540 agcggcggct ccgcgcaatg ggcggccagc atcggccacgacctggccag cctgggcggc 600 gatgacagca gtgcggcccc acgcattgcc aaccattacccggcgcttac cggccaattc 660 gtttcgttct cgatcctggt ttga 684 102 1593 DNAPseudomonas mendocina KR-1 102 atgaccgagc aaacccagaa caccctgattccccgtggcg tgaatgacgc caacctccag 60 caagccctgg ccaagttccg caagctgctgggcgaggaca acgtcctggt caaggacgag 120 caactcatcc cctacaacaa gatcatgatcgcagtggaca acgccgaaca cgcgccctcc 180 gctgctgtca ccgccaccac tgtggaacaggtgcagggcg tggtgaagat ctgcaacgaa 240 tacggcattc cggtgtggac catctccaccggccgcaact tcggttacgg ctcggcggcc 300 cccggccagc gtggccaggt gatcctcgacctgaagaaaa tgaacaagat catccacgta 360 gacccggacc tgtgcaccgc cctggtggaaccgggggtga cctaccagca gctgtacgat 420 tacctggaag agaacaacat cccgctgatgctgtccttct ctgcaccctc ggccatcgcc 480 ggcccgctgg gcaacaccat ggaccgtggcgtgggctaca ccccctacgg cgagcacttc 540 ctcatgcagt gcggcatgga agtggtgctggccaatggcg acgtctaccg caccggcatg 600 ggcggggtga aaggcgacaa cgcctggcaggtgttcaagt ggggctacgg cccgaccctg 660 gacggcatgt tcacccaggc caactacggcatctgcacca agatgggttt ctggctgatg 720 cccaagcccc cggtgttcaa gcccttcgagatcaagttcg agaacgagtc cgacatcagc 780 gagatcgtcg aattcatccg tccgctgcgcatcgcccagg tcatcccaaa ctccgtggtg 840 atcgccggtg tgctctggga ggcctccacctgcaataccc gccgctcgga ctacaccact 900 gagccgggcg ccactcccga caccatcctgaagcagatcc agaaggacaa ggaactcggc 960 gcctggaacg tctatgccgc tctctacggcacgcaggaac aggtggacgt gaactggaag 1020 atcgtcaccg gcgccctggc caaactgggcaagggcagga ttgtcaccca ggaagaggcc 1080 ggcgataccc agcccttcaa gtaccgttcccagttgatgt ccggcgtccc caacctgcag 1140 gaattcggcc tgtacaactg gcgcgggggcggcggctcca tgtggttcgc cccggtcagc 1200 caggcccgtg gcatcgagtg cgacaagcagcaggcgctgg ccaagaagat cctcaacaag 1260 cacggcctgg actacgtcgg cgagttcattgtcggctggc gcgacatgca ccacgtaatc 1320 gacgtgctgt acgaccgcac caaccccgaggaaacccaac gcgcctacgc ctgcttccac 1380 gagttgctgg atgagttcga gaagcacggctatgcggtgt accgcgtgaa cactgcgttc 1440 caggagcgcg tggcgcagag gtacggcacggtcaagcgca ggtggaacac gccatcaagc 1500 gcgccctgga cccgaacaac atcctggcacccggcaaatc cggcatcgac ctcgccaaca 1560 agttctaacc ctaagcaaga ccccgccgggtaa 1593 103 1371 DNA Pseudomonas mendocina KR-1 103 atgataaaaatgaaaattgc cagcgtactc gtactgcctt tgagcggtta tgcttttagc 60 gtgcacgctacacaggtgtt cgatctggag ggttatgggg caatctctcg tgccatggga 120 ggtaccagctcatcgtatta taccggcaat gctgcattga tcagcaaccc cgctacattg 180 agcttggctccggacggaag tcagtttgag ctcgggccgg atatagtaag taccgatatt 240 gaggttcgtgacagcagcgg tgcgaaagta aaaagcagca cggaatccaa taatcgaggc 300 ccctatatcggtccgcagtt gagctatgtt actcagctgg atgactggcg tttcggtgct 360 gggttgtttgtgagtagtgg gctgggtaca gagtatggaa gtaacagttt cttgtcacag 420 acagaaaatggcacccaaac cagctttgac aattccagcc gtctgattgt gttgcgcgct 480 cctgtaggctttagttatca agtaacacca caacttacag tcggcgcaag tgctgatctg 540 gtctggacctcactcaatct cgagcttcta ctcccatcat ctcaggtggg agcactcgct 600 gcgcagggtaatctttcagg tgatttagtc gccccactcg ctgggtttgt gggtgctggt 660 ggtgctgcacatttcagtct aagtcgcaac aacccagttg gcggtgccgt ggatgcaatc 720 gggtggggtgggcgtttggg tctgacctac aagctcacgg ataagacagt ccttggtgcg 780 atgtacaacttcaagacttc tgtgggcgac ctcgaaggga cggcaacact ttctgctatc 840 agcggtgatggtgcggtgtt gccattacat ggcgatatcc gcgtaaaaga cttcgagatg 900 cccgccagtctgacgttcgg ctttgctcat caattcaacg agcgttggct ggttgctgct 960 gatgtcaagcgtgtctactg gagcgatgtc atggaagaca tcagtgtgga tttcaaatcg 1020 cagtcaggtgggattgatat cgaattacca cacaactatc aggatattac ggtggcctcc 1080 atcggcaccgcttacagagt taatgacaag ctaactcttc gtgctggata tagctatgcg 1140 caacaggcgctggacagtag gctgatattg ccagtaattc cagcttattt gaagaaacac 1200 gtttctctcggtagcgatta tagttttgat aaaaaatcaa aactcaattt ggcgatttct 1260 tttggcctaaaagagagctt gaacacacca tcatacctaa gcggcaccga aacgttgaag 1320 caaagccacagccaaataaa cgcagtggtt tcctacagca aaagctttta a 1371 104 17 DNA ArtificialSequence Description of Artificial Sequence primer 104 gcttccacggtatctcg 17 105 17 DNA Artificial Sequence Description of ArtificialSequence primer 105 cagtcaatcc gctgcac 17 106 20 DNA Artificial SequenceDescription of Artificial Sequence primer 106 gcagtatggt cacctgttcc 20107 19 DNA Artificial Sequence Description of Artificial Sequence primer107 ggttcgacca ccaggctac 19 108 19 DNA Artificial Sequence primer 108ggatctcaaa gccctgacc 19 109 21 DNA Artificial Sequence Description ofArtificial Sequence primer 109 tgctgcacaa ggccggtatc g 21 110 21 DNAArtificial Sequence Description of Artificial Sequence primer 110ggtcatgaac cagctgaagc g 21 111 19 DNA Artificial Sequence Description ofArtificial Sequence primer 111 cctgtccgtt aatcgaacg 19 112 3554 DNAPseudomonas putida 112 atgagctcct tggatagaaa aaagcctcaa aatagatcgaaaaataatta ttataatatc 60 tgcctcaagg agaaaggatc tgaagagctg acgtgtgaagaacatgcacg catcatattt 120 gatgggctct acgagtttgt gggccttctt gatgctcatggaaatgtgct tgaagtgaac 180 caggtcgcat tggagggggg cgggattact ctggaagaaatacgagggaa gccattctgg 240 aaggcgcgtt ggtggcaaat ttcaaaaaaa accgaggcgacccaaaagcg acttgttgaa 300 actgcatcat ccggtgaatt tgttcgctgt gatgttgagattcttggaaa atcaggtgga 360 agagaggtaa tatcggtcga tttttcattg ctgccaatttgcaatgaaga agggagcatt 420 gtttaccttc ttgcggaagg gcgcaatatt accgataagaagaaagccga ggccatgctg 480 gcgttgaaga accaggaatt ggagcagtcg gttgagtgtatccgaaaact cgataatgcg 540 aagagtgatt tctttgccaa ggtgagccat gagttgcgcactccgctgtc tttgattcta 600 ggccactgga agccgttatg gcgggcagag gctgggcgtgaatcgccgta ttggaagcag 660 tttgaggtca ttcagcgtaa tgcaatgacc ctgttgaaacaggttaacac gctgcttgac 720 ttggcgaaaa tggacgcccg gcagatgggg ctttcctatcggcgagccaa tcttagtcag 780 ctcacccgta ctattagctc gaattttgaa ggaatagcccagcaaaaatc aataacgttc 840 gatacaaaac tgcctgtaca gatggtcgct gaggtggattgtgagaaata cgaacgcatt 900 atccttaact tgctttccaa tgcgtttaaa ttcacccctgacggggggct tatccgttgc 960 tgtcttagtt tgagtcgacc aaattatgcc ttggttactgtatctgatag cgggccgggt 1020 attcctcctg cactgcgtaa agaaatattt gaacgtttccaccagctaag ccaggaaggt 1080 caacaagcta cgcggggtac aggcttgggg ctttccattgtgaaagaatt cgttgaattg 1140 caccgtggaa caatttctgt aagtgatgcc ccgggcgggggggcgctttt tcaggtaaag 1200 ctgccgctga atgctcctga aggtgcttat gttgcgagtaacaccgcgcc gcgaagagat 1260 aatcctcagg tcgtggatac ggatgagtac cttttgctggcgcccaatgc ggaaaatgaa 1320 gccgaggtgc ttccatttca atccgaccag cctcgggtgctaatcgttga agataaccct 1380 gatatgcgtg gttttataaa ggactgtctc agtagcgactatcaagttta tgttgcaccc 1440 gacggtgcaa aggcattgga gttgatgtca aacatgccgccagacctgtt gattacagac 1500 ctgatgatgc ctgttatgag cggcgatatg ctggttcaccaagtgcgtaa gaaaaatgaa 1560 ctttcacata tcccgatcat ggtgctgtcg gccaagtcagacgcagaact gcgtgtgaaa 1620 ttgctctccg agtcggtgca ggactttctt cttaagccattttctgctca tgagctacga 1680 gcgcgtgtaa gcaatctggt atccatgaag gtggcaggcgatgcgttgcg taaggagctt 1740 tccgatcagg gggatgatat tgcgatactt actcaccgtctgatcaaaag tcgccatcgt 1800 cttcagcaga gtaacatcgc attatccgcc tcggaagcgcgttggaaagc agtgtatgaa 1860 aactctgcgg ccggtattgt actgaccgac ccggaaaaccgaatactcaa cgccaatcct 1920 gcatttcaac gcattaccgg atatggggaa aaggatttggagggactttc catggagcaa 1980 ttgactccat ctgacgaaag cccacagata aagcagcgtctggccaattt gcttcagggt 2040 gggggagcgg aatacagtgt ggagcgctcc tatctatgcaaaaatggttc tacgatttgg 2100 gccaatgcga gtgtctcgct gatgcctcaa cgtgtcggtgaatctccagt tatactgcag 2160 atcatcgatg acatcactga gaagaaacaa gcacaggaaaatcttaacca attgcagcaa 2220 caacttgtgt acgtttcccg atcagctacg atgggtgaatttgcagccta tattgcacac 2280 gagataaacc aaccgctctc ggcgatcatg accaatgccaatgctggcac acgttggtta 2340 ggtaatgagc catctaacat cccagaggct aaagaggcactggctcgcat tatccgagat 2400 tccgaccgcg ctgcagaaat tatccgtatg gtacgctccttcctgaagcg tcaagaaacg 2460 gtgctgaaac cgattgatct aaaagcactg gtaactgatacaagcctgat acttaaggcc 2520 cctagtcaga ataacagtgt caatttggat gttgttgcggatgatgaact ccctgagata 2580 tggggggatg gtgtacagat ccagcagttg ataataaatctggctatgaa cgctattgaa 2640 gcgatcagcc aagccgactg tgaaaccagg cagctaaccctgtcattctc aggcaatgat 2700 acaggtgatg cgcttgttat ctcagtgaaa gatacaggtccaggtatttc agagaggcag 2760 atggcgcagt tgttcaacgc attctacacc acaaaaaaagaagggcttgg tatgggattg 2820 gcaatctgtc ttacaatcac ggaagtgcat aacggtaaaatatgggttga gtgcccgccc 2880 gctgggggtg cttgtttcct ggtaagtatc cctgccagacagggctccgg cacatgagtg 2940 atcgggcatc tgttatctat atcctcgatg acgacaatgcagtactggaa gcactgagca 3000 gcttggtgcg ttcaatcggc ctgagtgtcg agtgtttttcatccgctagc gtattcctga 3060 acgatgtcaa tcgctctgcc tgtggctgtc taattttggatgtccgtatg cccgagatga 3120 gcgggttgga tgtgcaacga caactgaaag agcttggcgagcaaatcccc attattttta 3180 tcagcggcca cggtgatatt ccgatggcag tcaaagcgatcaaggcgggt gcggtagact 3240 tcttcactaa accttttcga gaagaggagc tgcttggcgctattcgcgcc gcgctgaagt 3300 tggcgcccca gcagagatca aacgctcccc gagtcagcgagcttaaagag aattacgaaa 3360 gcctcagcaa acgcgagcaa caggtgctta agttcgtcttgcgaggatat ctaaacaagc 3420 agacggctct agagcttgat atatcggaag caacagtgaaagtgcaccgc cataatatca 3480 tgaggaaaat gaaagtatct tcaatccagg atctggttcgagtaactgag cggctcaagg 3540 atagcctgga atag 3554

What is claimed is:
 1. An isolated nucleic acid fragment encoding abacterial toluene monooxygenase enzyme pathway selected from the groupconsisting of: (a) an isolated nucleic acid fragment encoding all or asubstantial portion of the amino acid sequence selected from the groupconsisting of SEQ ID NOs:2, 3, 4, 6 and 92; (b) an isolated nucleic acidfragment that is substantially similar to an isolated nucleic acidfragment encoding all or a substantial portion of the amino acidsequence selected from the group consisting of SEQ ID NOs:2, 3, 4, 6 and92; (c) an isolated nucleic acid fragment encoding a polypeptide of atleast 111 amino acids having at least 60% identity based on theSmith-Waterman method of alignment with the amino acid sequence selectedfrom the group consisting of SEQ ID NOs:2, 3, 4, 6 and 92; (d) anisolated nucleic acid fragment that hybridizes with (a) underhybridization conditions of 0.1× SSC, 0.1% SDS, 65° C. and washed with2× SSC, 0.1% SDS followed by 0.1× SSC, 0.1% SDS; and (e) an isolatednucleic acid fragment that is complementary to (a), (b), (c), or (d). 2.The isolated nucleic acid fragment of claim 1 selected from the groupconsisting of SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:102,and SEQ ID NO:103.
 3. A polypeptide encoded by the isolated nucleic acidfragment of claim
 1. 4. The polypeptide of claim 3 selected from thegroup consisting of SEQ ID NOs:2, 3, 4, 6 and
 92. 5. A chimeric genecomprising the isolated nucleic acid fragment of claim 1 operably linkedto suitable regulatory sequences.
 6. A host cell transformed with thechimeric gene of claim
 5. 7. The host cell of claim 6 wherein the hostcell is a bacterium.
 8. The transformed host cell of claim 7 wherein thehost cell is selected from the group consisting of Pseudomonas,Burkholderia, Acinetobacter, and Agrobacterium.
 9. A method of obtaininga nucleic acid fragment encoding all or a substantial portion of atoluene monooxygenase enzyme pathway comprising: (a) probing a genomiclibrary with the nucleic acid fragment of claim 1; (b) selecting for aDNA clone that hybridizes with the nucleic acid fragment of claim 1; and(c) sequencing the genomic fragment that comprises the clone identifiedin step (b), wherein the sequenced genomic fragment encodes all orsubstantially all of the amino acid sequence encoding a toluenemonooxygenase enzyme pathway.
 10. A method of obtaining a nucleic acidfragment encoding all or a substantial portion of the bacterial toluenemonooxygenase enzyme pathway comprising: (a) synthesizing at least oneoligonucleotide primer corresponding to at least a portion of thesequence selected from the group consisting of SEQ ID NOs:2, 3, 4, 6 and92; and (b) amplifying an insert present in a cloning vector using theoligonucleotide primer of step (a), wherein the amplified insert encodesa portion of an amino acid sequence encoding a bacterial toluenemonooxygenase enzyme pathway.
 11. The product of the method of claims 9or
 10. 12. A mutated bacterial toluene monooxygenase gene encoding aprotein having an altered biological activity produced by a methodcomprising the steps of: (i) contacting a mixture of nucleotidesequences with restriction endonucleases, the mixture comprising: (a)one or more native bacterial toluene monooxygenase enzyme pathway genesselected from the group consisting of SEQ ID NOs:98, 99, 100, 102, and103; (b) a first population of nucleotide fragments that hybridize tothe at least one native bacterial toluene monooxygenase enzyme pathwaygene sequence of (a); and (c) a second population of nucleotidefragments that will not hybridize at high stringency to the nativebacterial toluene monooxygenase enzyme pathway gene sequence of (a);(ii) denaturing the mixture of restriction fragments produced in (i);(iii) incubating the denatured mixture of restriction fragments of step(ii) with a polymerase; (iv) optionally adding a ligase to the denaturedmixture of (iii); (v) repeating steps (ii) and (iii) at least one time;and (vi) screening the products of (v) to identify a mutated bacterialtoluene monooxygenase enzyme pathway gene encoding a protein having analtered biological activity relative to the native bacterial toluenemonooxygenase enzyme pathway gene.
 13. A method for the production ofp-hydroxybenzoate comprising: (i) contacting a transformed host cellwith a medium containing (a) an aromatic organic substrate selected fromthe group consisting of p-cresol, p-hydroxybenzyl alcohol,p-hydroxybenzaldehyde, and aromatic compounds degradated by the toluenemonooxygenase enzyme pathway, (b) at least one fermentable carbonsubstrate, and (c) a nitrogen source, the transformed cell 1) lacking ap-hydroxybenzoate hydroxylase activity and 2) comprising genes encodingPcuR, p-cresol methylhydroxylase, and p-hydroxybenzoate dehydrogenaseactivities, each gene operably linked to suitable regulatory sequences;(ii) incubating the transformed host cell for a time sufficient toproduce PHBA; and (iii) optionally recovering the p-hydroxybenzoateproduced in (ii).
 14. A method for the production of p-hydroxybenzoatecomprising: (i) contacting a transformed host cell with a mediumcontaining (a) an aromatic organic substrate toluene, p-cresol,p-hydroxybenzyl alcohol, p-hydroxybenzaldehyde, and aromatic compoundsdegradated by the toluene monooxygenase enzyme pathway, (b) at least onefermentable carbon substrate, and (c) a nitrogen source, the transformedcell 1) lacking a p-hydroxybenzoate hydroxylase activity 2) andcomprising genes encoding toluene-4-monooxygenase, TmoX, PcuR, p-cresolmethylhydroxylase, and p-hydroxybenzoate dehydrogenase activities, eachgene operably linked to suitable regulatory sequences; (ii) incubatingthe transformed host cell for a time sufficient to producep-hydroxybenzoate; and (iii) optionally recovering the p-hydroxybenzoateproduced in (ii).
 15. The method of claim 13 wherein the aromaticorganic substrate is p-cresol.
 16. The method of claim 14 wherein thearomatic organic substrate is toluene.
 17. The method of claim 13 or 14wherein the fermentable carbon substrate is selected from the groupconsisting of monosaccharides, oligosaccharides, polysaccharides, carbondioxide, methanol, formaldehyde, formate, and carbon-containing amines.18. The method of claim 17 wherein the fermentable carbon substrate isglucose.
 19. The method of claim 13 or 14 wherein the transformed hostcell is selected from the group consisting of Pseudomonas, Burkholderia,Acinetobacter, and Agrobacterium.
 20. The method of claim 15 whereinp-cresol is present in the medium in a concentration of less than 500ppm.
 21. The method of claim 15 wherein p-cresol is present in themedium from about 30 ppm to about 60 ppm.
 22. The method of claim 16wherein toluene is present in the medium in a concentration of less than500 ppm.
 23. The method of claim 16 wherein toluene is present in themedium from about 30 ppm to about 60 ppm.
 24. An expression plasmid pMC4as shown in FIG.
 4. 25. The method of claim 14 wherein the transformedhost cell comprises plasmid pMC4 as shown in FIG.
 4. 26. The method ofclaim 14 wherein the tranformed host cell further comprises the genesencoding TodST activity.